Suppression of cancer metastasis

ABSTRACT

Methods are provided for suppressing cancer metastasis. Cancer metastasis is the most common cause of treatment failure and death in cancer patients. Tumor cell invasion and/or migration can be significantly inhibited after fibrillar proteins (rVP1, F-HSA, and F-BSA) treatment in vitro. In addition, rVP1 can significantly suppress murine and human breast cancer metastasis and human prostate and ovarian cancer metastasis in vivo while F-HSA can significantly suppress murine breast cancer metastasis. Compositions of fibrillar proteins as anti-cancer metastasis therapeutics and methods of use thereof are provided herein.

INTRODUCTION

Cancer metastasis is a process that involves a series of sequentialsteps and requires a cascade of host-tumor cell interactions (Steeg P Set al. (2007) Nature 449:671-3). These steps include detachment from theprimary tumor, invasion into and arrest in circulatory systems,extravasation into the parenchyma of organs; and proliferation inassociation with angiogenesis (Sawyer T K et al. (2004) Expert OpinInvestig Drugs 13:1-19). There are growing interests in investigatingthe mechanisms of migration and invasion, which are unveiled to be acritical step of the metastatic process. Interference at any one ofthese steps to block the metastatic cascade represents an attractiveapproach to prevent the formation of metastatic tumor growths.

Our previous data showed that recombinant capsid protein VP1 (rVP1) offoot-and-mouth disease virus (FMDV), induced apoptosis in several kindsof cancer cells via integrin signaling pathway (Peng J M et al. (2004)J. Biol. Chem. 279:52168-74). It was discovered that using the sameprocess for refolding rVP1 into water-soluble form, globular bovineserum albumin (G-BSA) can be converted into fibrillar BSA (F-BSA), whichlike rVP1, induced tumor cell apoptosis via integrin/FAK/Akt signalingpathway (Huang et al. (2009) BMC Biotechnol. 9:2).

SUMMARY OF THE INVENTION

Methods are provided for suppressing cancer metastasis. Cancermetastasis is the most common cause of treatment failure and death incancer patients. Tumor cell invasion and/or migration is significantlyinhibited after contacting the tumor cells with fibrillar proteins,which fibrillar proteins include, without limitation, rVP1, F-HSA, andF-BSA. Tumor cells of interest include carcinomas, for example breastcarcinoma, epithelial adenocarcinoma of the ovary, adenocarcinoma of theprostate, etc.

In one aspect, the invention relates to a composition comprising atherapeutically effective amount of fibrillar human serum albumin and apharmaceutically acceptable carrier for use in treating a mammal havingcancer, e.g. for suppressing cancer cells, suppressing metastasis, etc.

In some embodiments of the invention, tumor cells are contacted in vivowith fibrillar proteins, which contacting may be local, e.g.intratumoral introduction or injection, or systemic. For example rVP1 isshown herein to significantly suppress murine and human breast cancermetastasis and human prostate and ovarian cancer metastasis in vivo.F-HSA is shown to significantly suppress breast cancer metastasis. Inone embodiment, the invention relates to a composition as aforementionedfor use in treating cancer in a mammal, wherein the cancer is at leastone chosen from breast cancer, ovarian cancer, cervical cancer, prostatecancer, and lung cancer.

In some embodiments of the invention compositions of fibrillar proteinsas anti-cancer metastasis therapeutics are provided, where thecomposition provides for a pharmaceutical formulation in a doseeffective to inhibit metastasis. In another aspect, the inventionrelates to a method comprising manufacturing a composition for use intreating a patient having cancer. The method comprises manufacturingfibrillar human serum albumin, and mixing the fibrillar human serumalbumin with a pharmaceutically acceptable carrier. In another aspect,the invention relates to a method comprising dissolving HSA in an SDSsolution; applying the dissolved HSA through a gel filtration columnwith a pore size to separate proteins of 70 kDa molecular weight andabove; eluting the HSA from the column; and dialyzing the solutionagainst phosphate buffered saline to remove the SDS.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. The effects of rVP1 on cell invasion/migration and cytotoxicityin MDA-MB-231 cells, PC-3 cell, and 22Rv1 cells. (A&B) Cellinvasion/migration of MDA-MB-231 cells, PC-3 cell, and 22Rv1 cells withrVP1 treatment for 24 hrs was measured by using Boyden chamber assay.rVP1 significantly suppressed tumor cell invasion/migration (C) Thecytotoxicity of MDA-MB-231 cells, PC-3 cell, and 22Rv1 cells with rVP1treatment for 24 hrs was measured by using MTT assay. 0.1 μM and 0.2 μMrVP1 did not affect tumor cell viability. Data represent means±S.D.(n=3). *: P<0.05, **: P<0.01 and ***: P<0.001 were relative to control(0 μM rVP1 treatment).

FIG. 2. The effects of rVP1 on cell invasion and cytotoxicity in SK-OV-3cells and CaSki cells. (A) Cell invasion of SK-OV-3 cells and CaSkicells with rVP1 treatment for 24 hrs was measured by using Boydenchamber assay. rVP1 significantly suppressed tumor cell invasion (B)Cell cytotoxicity of SK-OV-3 cells and CaSki cells with rVP1 treatmentfor 24 hrs was measured by using MTT assay. 0.2 μM to 0.4 μM rVP1 inSKOV-3 cells and 0.2 μM to 0.6 μM rVP1 in CaSki cells did not affectcell viability. White bar, SKOV-3 cells; black bar, CaSki cells. Datarepresent means±S.D. (n=3). *: P<0.05; **: P<0.01; ***: P<0.001.

FIG. 3. The effects of rVP1 on cell invasion and cytotoxicity inSK-OV-3ip.1 cells. (A) SK-OV-3ip.1 cells were plated onto the upperchamber for 1 hour followed by rVP1 treatment for 24 h. Afterincubation, cells from the lower membrane surface were dissociated andcounted. (B) SK-OV-3ip.1 cells were treated with rVP1 for 24 h thenWST-1 assays were performed. Cell survival rates were determined bymeasuring the absorbance at 450 nm (690 nm as reference). The percentageof cell survival was calculated as (O.D_(treatment)/O.D_(control))×100%.Data represent means±S.D. (n=3). *, P<0.05 and **, P<0.01 were relativeto control.

FIG. 4. The metastatic ability of MDA-MB-231 cells was lost after rVP1treatment in vivo. 0.1 μM and 0.2 μM rVP1-treated MDA-MB-231 cells invitro for 24 hrs then harvested and intravenously injected in micethrough tail vein. After 14 days, sacrifice was performed. The grossappearance (A) and histopathology examination (B and C) of lung in threedifferent groups of mice were measured. rVP1 significantly inhibitedmetastatic ability of MDA-MB-231 cells. ***: P<0.001.

FIG. 5. rVP1 suppressed PC-3 cells metastasized to lymph nodes andinhibited osteolysis in pelvic bones. (A) The lymph nodes of normal andtumor bearing mice with or without rVP1 treatment. (B) The pelvic bonesof normal and tumor bearing mice with or without rVP1 treatment (arrow).

FIG. 6. In vivo histological appearance of liver tumor metastasis fromSK-OV-3 implanted nude mice were prevented by rVP1. Representative liversections with H&E stain from rVP1 treated and nontreated mice.BALB/cAnN-Foxn1 female nude mice were implanted with 5×10⁶ SK-OV-3cancer cells per mouse by intraperitoneal injection. (a-b) After 60days, liver from PBS-treated SK-OV-3 bearing mice were surrounded bytumor cells. (c-d) 340 days after SK-OV-3 implantation, liver fromrVP1-treated mice did not show apparent invasion. (e-f) Liver fromnormal mouse (i.e., without SK-OV-3 implantation).

FIG. 7. The effects of F-HSA on cell migration, invasion andcytotoxicity in MDA-MB-231 cells, PC-3 cells, 22Rv1 cells, and CaSkicells. In vitro migration and invasion assay, F-HSA significantlyinhibited cell invasion (black bar) and migration (white bar) inMDA-MB-231 cells (A), PC-3 cells (C), 22Rv1 cells (E), and CaSki cells(G) by using Boyden chamber assay. The effects of F-HSA on cytotoxicityin MDA-MB-231 cells, PC-3 cells, 22Rv1 cells, and CaSki cells by usingMTT assay. (B, D, F, and H). Migration and invasion were represented aspercentage of the number of treated cells to that of untreated cells.Shown were the means±S.D. of three independent experiments with threereplicates in each. **: P<0.01 and ***: P<0.001 were relative tountreated cells.

FIG. 8. F-HSA suppressed TS/A tumor cell metastasis in vivo. (A) Thegross appearance and histopathology examination of lung from mice withor without F-HSA treatment. (B & C) The related lung weight and thenumber of tumor foci in lung from mice with or without F-HSA treatment.***: P<0.001.

FIG. 9. F-HSA suppressed MDA-MB-231 tumor cell metastasis in vivo. (A)The gross appearance and histopathology examination of lung from micewith or without F-HSA treatment. (B & C) The related lung weight and thenumber of tumor foci in lung with or without F-HSA treatment. ***:P<0.001.

FIG. 10. The effects of F-BSA on cell invasion and cytotoxicity in CaSkicells. (A) Cell invasion of CaSki cells treated with F-BSA for 24 hrswas measured by Boyden chamber assay. F-HSA significantly suppressedtumor cell invasion. (B) The cytotoxicity of CaSki cells treated withF-BSA for 24 hrs was measured by MTT assay. F-HSA at 0.1 μM or 0.2 μMconcentration had no effect on the cell viability of CaSki cells. Datarepresent means±S.D. (n=3). *: P<0.05; **: P<0.01.

FIG. 11. A schedule of the tumor implantation with rVP1 treatment.

FIG. 12 is an implementation of experimental data showing a comparisonof fluorescence level of increasing concentrations of F-HSA, HSA, and Aβ (1-42) after incubating with 20 μM amyloid-specific dye ThT for 1 h.

FIG. 13 is an implementation of experimental data showing the cytotoxiceffects of F-HSA on breast cancer cells.

FIG. 14 is an implementation of experimental data showing the cytotoxiceffects of F-HSA on breast cancer cells.

FIG. 15 is an implementation of experimental data showing the cytotoxiceffects of F-HSA on ovarian cells.

FIG. 16 is an implementation of experimental data showing the cytotoxiceffects of F-HSA on cervical cancer cells.

FIG. 17 is an implementation of experimental data showing the cytotoxiceffects of F-HSA on prostate cancer cells.

FIG. 18 is an implementation of experimental data showing the cytotoxiceffects of F-HSA on prostate cancer cells.

FIG. 19 is an implementation of experimental data showing the cytotoxiceffects of F-HSA on lung cancer cells.

FIG. 20A-20H are implementations of experimental data showing the effectof F-HSA in reducing tumor cell migrations and invasion withouteffecting viability of normal cells.

FIG. 21A-21C are implementations of experimental data showing the effectof F-HSA in suppressing the metastasis of mouse breast tumor TS/A cellsto the lung.

FIG. 22A-22C are implementations of experimental data showing the effectof F-HSA in suppressing the metastasis of mouse breast tumor MDA-MB-231cells to the lung.

DEFINITIONS

In the description that follows, a number of terms conventionally usedin the field of cell culture are utilized extensively. In order toprovide a clear and consistent understanding of the specification andclaims, and the scope to be given to such terms, the followingdefinitions are provided.

The terms “subject,” “individual,” and “patient” are usedinterchangeably herein to refer to a mammal being assessed for treatmentand/or being treated. In an embodiment, the mammal is a human. The terms“subject,” “individual,” and “patient” thus encompass individuals havingcancer (e.g., colorectal cancer, adenocarcinoma of the ovary orprostate, breast carcinoma, etc.), including those who have undergone orare candidates for resection (surgery) to remove cancerous tissue (e.g.,cancerous colorectal tissue). Subjects may be human, but also includeother mammals, particularly those mammals useful as laboratory modelsfor human disease, e.g. mouse, rat, etc.

As used herein, the terms “treatment,” “treating,” and the like, referto administering an agent, or carrying out a procedure (e.g., radiation,a surgical procedure, etc.), for the purposes of obtaining an effect.The effect may be prophylactic in terms of completely or partiallypreventing a disease or symptom thereof and/or may be therapeutic interms of effecting a partial or complete cure for a disease and/orsymptoms of the disease. “Treatment,” as used herein, covers anytreatment of any metastatic tumor in a mammal, particularly in a human,and includes: (a) preventing the disease or a symptom of a disease fromoccurring in a subject which may be predisposed to the disease but hasnot yet been diagnosed as having it (e.g., including diseases that maybe associated with or caused by a primary disease; (b) inhibiting thedisease, i.e., arresting its development; and (c) relieving the disease,i.e., causing regression of the disease. In tumor (e.g., cancer)treatment, a therapeutic agent may directly decrease the metastasis oftumor cells.

The term “cell culture” or “culture” means the maintenance of cells inan artificial, in vitro environment. It is to be understood, however,that the term “cell culture” is a generic term and may be used toencompass the cultivation not only of individual cells, but also oftissues or organs.

The term “tumor,” as used herein, refers to all neoplastic cell growthand proliferation, whether malignant or benign, and all pre-cancerousand cancerous cells and tissues.

The terms “cancer,” “neoplasm,” and “tumor” are used interchangeablyherein to refer to cells which exhibit autonomous, unregulated growth,such that they exhibit an aberrant growth phenotype characterized by asignificant loss of control over cell proliferation. In general, cellsof interest for detection, analysis, classification, or treatment in thepresent application include precancerous (e.g., benign), malignant,pre-metastatic, metastatic, and non-metastatic cells. Examples of cancerinclude but are not limited to, breast cancer, colon cancer, lungcancer, prostate cancer, hepatocellular cancer, gastric cancer,pancreatic cancer, cervical cancer, ovarian cancer, liver cancer,bladder cancer, cancer of the urinary tract, thyroid cancer, renalcancer, carcinoma, melanoma, head and neck cancer, and brain cancer.

Depending on the nature of the cancer, an appropriate patient sample isobtained. As used herein, the phrase “cancerous tissue sample” refers toany cells obtained from a cancerous tumor. In the case of solid tumorswhich have not metastasized, a tissue sample from the surgically removedtumor will typically be obtained and prepared for testing byconventional techniques. Alternatively, a body fluid sample, such aslymph, blood or serum sample, or an exudate fluid sample such as thecancerous organ exudate (e.g., exudate from the breast) may be collectedand used as the sample to be analyzed. In the case of leukemias,lymphocytes or leukemic cells will be obtained and appropriatelyprepared. Similarly, in the case of any metastasized cancer, cells maybe drawn from a body fluid such as lymphatic fluid, blood, serum, or adistally infected organ or exudate thereof.

As used herein, the term “metastasis” refers to the growth of acancerous tumor in an organ or body part, which is not directlyconnected to the organ of the original cancerous tumor. Metastasis willbe understood to include micrometastasis, which is the presence of anundetectable amount of cancerous cells in an organ or body part which isnot directly connected to the organ of the original cancerous tumor.Metastasis can also be defined as several steps of a process, such asthe departure of cancer cells from an original tumor site, and migrationand/or invasion of cancer cells to other parts of the body. Therefore,the present invention contemplates a method of determining the risk offurther growth of one or more cancerous tumors in an organ or body partwhich is not directly connected to the organ of the original canceroustumor and/or any steps in a process leading up to that growth.

The “pathology” of cancer includes all phenomena that compromise thewell-being of the patient. This includes, without limitation, abnormalor uncontrollable cell growth, metastasis, interference with the normalfunctioning of neighboring cells, release of cytokines or othersecretory products at abnormal levels, suppression or aggravation ofinflammatory or immunological response, neoplasia, premalignancy,malignancy, invasion of surrounding or distant tissues or organs, suchas lymph nodes, etc.

As used herein, the terms “cancer recurrence” and “tumor recurrence,”and grammatical variants thereof, refer to further growth of neoplasticor cancerous cells after diagnosis of cancer. Particularly, recurrencemay occur when further cancerous cell growth occurs in the canceroustissue. “Tumor spread,” similarly, occurs when the cells of a tumordisseminate into local or distant tissues and organs; therefore tumorspread encompasses tumor metastasis.

The term “diagnosis” is used herein to refer to the identification of amolecular or pathological state, disease or condition, such as theidentification of a molecular subtype of breast cancer, prostate cancer,or other type of cancer.

The term “prognosis” is used herein to refer to the prediction of thelikelihood of cancer-attributable death or progression, includingrecurrence, metastatic spread, and drug resistance, of a neoplasticdisease, such as lung, colon, skin or esophageal cancer. The term“prediction” is used herein to refer to the act of foretelling orestimating, based on observation, experience, or scientific reasoning.In one example, a physician may predict the likelihood that a patientwill survive, following surgical removal of a primary tumor and/orchemotherapy for a certain period of time without cancer recurrence.

As used herein, the phrase “disease-free survival,” refers to the lackof such tumor recurrence and/or spread and the fate of a patient afterdiagnosis, with respect to the effects of the cancer on the life-span ofthe patient. The phrase “overall survival” refers to the fate of thepatient after diagnosis, despite the possibility that the cause of deathin a patient is not directly due to the effects of the cancer. Thephrases, “likelihood of disease-free survival”, “risk of recurrence” andvariants thereof, refer to the probability of tumor recurrence or spreadin a patient subsequent to diagnosis of cancer, wherein the probabilityis determined according to the process of the invention.

As used herein, the term “correlates,” or “correlates with,” and liketerms, refers to a statistical association between instances of twoevents, where events include numbers, data sets, and the like. Forexample, when the events involve numbers, a positive correlation (alsoreferred to herein as a “direct correlation”) means that as oneincreases, the other increases as well. A negative correlation (alsoreferred to herein as an “inverse correlation”) means that as oneincreases, the other decreases.

The term “isolated” is intended to mean that a compound is separatedfrom all or some of the components that accompany it in nature.“Isolated” also refers to the state of a compound (e.g. protein)separated from all or some of the components that accompany it duringmanufacture (e.g., chemical synthesis, recombinant expression, culturemedium, and the like).

A “biological sample” encompasses a variety of sample types obtainedfrom an individual. The definition encompasses blood and other liquidsamples of biological origin, solid tissue samples such as a biopsyspecimen or tissue cultures or cells derived therefrom and the progenythereof. The definition also includes samples that have been manipulatedin any way after their procurement, such as by treatment with reagents;washed; or enrichment for certain cell populations, such as cancercells. The definition also includes sample that have been enriched forparticular types of molecules, e.g., nucleic acids, polypeptides, etc.The term “biological sample” encompasses a clinical sample, and alsoincludes tissue obtained by surgical resection, tissue obtained bybiopsy, cells in culture, cell supernatants, cell lysates, tissuesamples, organs, bone marrow, blood, plasma, serum, and the like. A“biological sample” includes a sample obtained from a patient's cancercell, e.g., a sample comprising polynucleotides and/or polypeptides thatis obtained from a patient's cancer cell (e.g., a cell lysate or othercell extract comprising polynucleotides and/or polypeptides); and asample comprising cancer cells from a patient. A biological samplecomprising a cancer cell from a patient can also include non-cancerouscells.

DESCRIPTION OF THE SPECIFIC EMBODIMENTS

The present disclosure relates to a process of producing fibrillarproteins and methods of treatment using fibrillar proteins. The methodof treatment involves suppression of cancer metastasis.

Method of Treatment

A method is disclosed herein for inhibiting cancer metastasis. Themethod involves administering a therapeutically effective amount of thefibrillar structure protein to a patient in need thereof. The method canfurther involve the steps of providing a protein, and changing theprotein into a fibrillar structure prior to administering the fibrillarprotein. Administration of the fibrillar protein inhibits tumor cellinvasion (e.g. into surrounding tissues) and/or migration.

The present method finds use in a variety of cancer therapies (includingcancer prevention and post-diagnosis cancer therapy) in a mammaliansubject, particularly in a human. Subjects having, suspected of having,or at risk of developing a tumor are contemplated for therapy describedherein.

Anti-cancer therapies in accordance with the subject method can beparticularly directed to cancerous cells that are metastatic or at ahigh risk of becoming metastatic. As such, fibrillar protein can be usedtherapeutically to effect/prevent adhesion and invasion of cancer cellsin other tissues.

For example, the cancer metastases that can be inhibited by the methodof the present disclosure include, but are not limited to, carcinomas,including adenocarcinomas, and particularly breast carcinomas,adenocarcinoma of the prostate and adenocarcinoma of the ovary. Othermetastases that can be treated include those that originate fromcancerous growth in kidney, lung, liver, skin (e.g. melanoma), colon,pancreas, or cervix.

The fibrillar structure protein used to treat the cancer can be analbumin, fibronectin, rVP1, rVP2, rVP3, P1, or chimeric proteincomprising parts from VP1, VP2, VP3, and/or VP4. Albumin proteins may beobtained from any animal of interest, e.g. human serum albumin, bovineserum albumin, etc. In certain embodiments, the fibrillar proteinadditionally induces cancer cell apoptosis by modulating the Aktsignaling pathway. In some instances, the fibrillar protein modulatesintegrin α 5β 1 or αv β 3 which leads to the deactivation of Akt. Inother instances, fibrillar albumin binds to integrin and causes cellularapoptosis mainly through the integrin/FAK/Akt/GSK-3β/caspase-3 pathway.

As noted above, the present method involves administering fibrillarproteins to a subject (e.g. a human patient) to, for example, tosuppress invasion and migration of cancerous cells. This can beaccomplished by administering a fibrillar protein to the subject asdescribed herein so as to provide for a decrease in the metastasis of acancer as compared to subjects who are not administered the fibrillarprotein. Therapies in accordance with the subject methods can also beuseful in preventing relapse, reducing migration of cancer cells,reducing tumor size, reducing tumor load, and/or improving the clinicaloutcome in patients.

Types of Cancer

The methods relating to cancer contemplated herein include, for example,use of fibrillar protein therapy as an anti-cancer metastasis therapy.The methods are useful in the context of treating or preventing a widevariety of cancers, such as cancers that can metastasize (e.g.carcinomas and sarcomas).

Carcinomas that can be amenable to therapy by a method disclosed hereininclude, but are not limited to, esophageal carcinoma, hepatocellularcarcinoma, basal cell carcinoma (a form of skin cancer), squamous cellcarcinoma (various tissues), bladder carcinoma, including transitionalcell carcinoma (a malignant neoplasm of the bladder), bronchogeniccarcinoma, colon carcinoma, colorectal carcinoma, gastric carcinoma,lung carcinoma, including small cell carcinoma and non-small cellcarcinoma of the lung, adrenocortical carcinoma, thyroid carcinoma,pancreatic carcinoma, breast carcinoma, ovarian carcinoma, prostatecarcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous glandcarcinoma, papillary carcinoma, papillary adenocarcinoma,cystadenocarcinoma, medullary carcinoma, renal cell carcinoma, ductalcarcinoma in situ or bile duct carcinoma, choriocarcinoma, seminoma,embryonal carcinoma, Wilm's tumor, cervical carcinoma, uterinecarcinoma, testicular carcinoma, osteogenic carcinoma, epitheliealcarcinoma, and nasopharyngeal carcinoma.

Sarcomas that can be amenable to therapy by a method disclosed hereininclude, but are not limited to, fibrosarcoma, myxosarcoma, liposarcoma,chondrosarcoma, chordoma, osteogenic sarcoma, osteosarcoma,angiosarcoma, endotheliosarcoma, lymphangiosarcoma,lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's sarcoma,leiomyosarcoma, rhabdomyosarcoma, and other soft tissue sarcomas.

Other solid tumors that can be amenable to therapy by a method disclosedherein include, but are not limited to, glioma, astrocytoma,medulloblastoma, craniopharyngioma, ependymoma, pinealoma,hemangioblastoma, acoustic neuroma, oligodendroglioma, menangioma,melanoma, neuroblastoma, and retinoblastoma.

Other cancers that can be amenable to treatment according to the methodsdisclosed herein include atypical meningioma (brain), islet cellcarcinoma (pancreas), medullary carcinoma (thyroid), mesenchymoma(intestine), hepatocellular carcinoma (liver), hepatoblastoma (liver),clear cell carcinoma (kidney), and neurofibroma mediastinum.

Further exemplary cancers that can be amenable to treatment using amethods disclosed herein include, but are not limited to, cancers ofneuroectodermal and epithelial origin. Examples of cancers ofneuroectodermal origin include, but are not limited to, Ewings sarcoma,spinal tumors, brain tumors, supratenbrial primative neuroectodermaltumors of infancy, tubulocystic carcinoma, mucinous tubular and spindlecell carcinoma, renal tumors, mediastinum tumors, neurogliomas,neuroblastomas, and sarcomas in adolescents and young adults. Examplesof epithelial origin include, but are not limited to, small cell lungcancer, cancers of the breast, eye lens, colon, pancreas, kidney, liver,ovary, and bronchial epithelium.

Combinations with Other Cancer Therapies

Therapeutic administration of the fibrillar protein can includeadministration as a part of a therapeutic regimen that may or may not bein conjunction with additional standard anti-cancer therapeutics,including but not limited to immunotherapy, chemotherapeutic agents andsurgery (e.g., as those described further below).

In addition, therapeutic administration of the fibrillar protein canalso be post-therapeutic treatment of the subject with an anti-cancertherapy, where the anti-cancer therapy can be, for example, surgery,radiation therapy, administration of chemotherapeutic agents, and thelike. Cancer therapy using fibrillar proteins of the present disclosurecan also be used in combination with immunotherapy. In other examples,the fibrillar proteins can be administered in combination with one ormore chemotherapeutic agents (e.g., cyclophosphamide, doxorubicin,vincristine and prednisone (CHOP)), and/or in combination with radiationtreatment and/or in combination with surgical intervention (e.g., pre-or post-surgery to remove a tumor). Where the fibrillar proteins areused in connection with surgical intervention, the fibrillar protein canbe administered prior to, at the time of, or after surgery to removecancerous cells, and may be administered systemically or locally at thesurgical site. The fibrillar protein alone or in combinations describedabove can be administered systemically (e.g., by parenteraladministration, e.g., by an intravenous route) or locally (e.g., at alocal tumor site, e.g., by intratumoral administration (e.g., into asolid tumor, into an involved lymph node in a lymphoma or leukemia),administration into a blood vessel supplying a solid tumor, etc.).

Any of a wide variety of cancer therapies can be used in combinationwith the fibrillar protein therapies described herein. Such cancertherapies include surgery (e.g., surgical removal of cancerous tissue),radiation therapy, bone marrow transplantation, chemotherapeutictreatment, biological response modifier treatment, and certaincombinations of the foregoing.

Radiation therapy includes, but is not limited to, X-rays or gamma raysthat are delivered from either an externally applied source such as abeam, or by implantation of small radioactive sources.

Chemotherapeutic agents are non-peptidic (i.e., non-proteinaceous)compounds that reduce proliferation of cancer cells, and encompasscytotoxic agents and cytostatic agents. Non-limiting examples ofchemotherapeutic agents include alkylating agents, nitrosoureas,antimetabolites, antitumor antibiotics, plant (vinca) alkaloids, andsteroid hormones.

Agents that act to reduce cellular proliferation are known in the artand widely used. Such agents include alkylating agents, such as nitrogenmustards, nitrosoureas, ethylenimine derivatives, alkyl sulfonates, andtriazenes, including, but not limited to, mechlorethamine,cyclophosphamide (CYTOXAN™), melphalan (L-sarcolysin), carmustine(BCNU), lomustine (CCNU), semustine (methyl-CCNU), streptozocin,chlorozotocin, uracil mustard, chlormethine, ifosfamide, chlorambucil,pipobroman, triethylenemelamine, triethylenethiophosphoramine, busulfan,dacarbazine, and temozolomide.

Antimetabolite agents include folic acid analogs, pyrimidine analogs,purine analogs, and adenosine deaminase inhibitors, including, but notlimited to, cytarabine (CYTOSAR-U), cytosine arabinoside, fluorouracil(5-FU), floxuridine (FudR), 6-thioguanine, 6-mercaptopurine (6-MP),pentostatin, 5-fluorouracil (5-FU), methotrexate,10-propargyl-5,8-dideazafolate (PDDF, CB3717),5,8-dideazatetrahydrofolic acid (DDATHF), leucovorin, fludarabinephosphate, pentostatine, and gemcitabine.

Suitable natural products and their derivatives, (e.g., vinca alkaloids,antitumor antibiotics, enzymes, lymphokines, and epipodophyllotoxins),include, but are not limited to, Ara-C, paclitaxel (TAXOL®), docetaxel(TAXOTERE®), deoxycoformycin, mitomycin-C, L-asparaginase, azathioprine;brequinar; alkaloids, e.g. vincristine, vinblastine, vinorelbine,vindesine, etc.; podophyllotoxins, e.g. etoposide, teniposide, etc.;antibiotics, e.g. anthracycline, daunorubicin hydrochloride (daunomycin,rubidomycin, cerubidine), idarubicin, doxorubicin, epirubicin andmorpholino derivatives, etc.; phenoxizone biscyclopeptides, e.g.dactinomycin; basic glycopeptides, e.g. bleomycin; anthraquinoneglycosides, e.g. plicamycin (mithramycin); anthracenediones, e.g.mitoxantrone; azirinopyrrolo indolediones, e.g. mitomycin; macrocyclicimmunosuppressants, e.g. cyclosporine, FK-506 (tacrolimus, prograf),rapamycin, etc.; and the like.

Other anti-proliferative cytotoxic agents are navelbene, CPT-11,anastrazole, letrazole, capecitabine, reloxafine, cyclophosphamide,ifosamide, and droloxafine.

Microtubule affecting agents that have antiproliferative activity arealso suitable for use and include, but are not limited to,allocolchicine (NSC 406042), Halichondrin B (NSC 609395), colchicine(NSC 757), colchicine derivatives (e.g., NSC 33410), dolstatin 10 (NSC376128), maytansine (NSC 153858), rhizoxin (NSC 332598), paclitaxel(TAXOL®), TAXOL® derivatives, docetaxel (TAXOTERE®), thiocolchicine (NSC361792), trityl cysterin, vinblastine sulfate, vincristine sulfate,natural and synthetic epothilones including but not limited to,eopthilone A, epothilone B, discodermolide; estramustine, nocodazole,and the like.

Hormone modulators and steroids (including synthetic analogs) that aresuitable for use include, but are not limited to, adrenocorticosteroids,e.g. prednisone, dexamethasone, etc.; estrogens and pregestins, e.g.hydroxyprogesterone caproate, medroxyprogesterone acetate, megestrolacetate, estradiol, clomiphene, tamoxifen; etc.; and adrenocorticalsuppressants, e.g. aminoglutethimide; 17α-ethinylestradiol;diethylstilbestrol, testosterone, fluoxymesterone, dromostanolonepropionate, testolactone, methylprednisolone, methyl-testosterone,prednisolone, triamcinolone, chlorotrianisene, hydroxyprogesterone,aminoglutethimide, estramustine, medroxyprogesterone acetate,leuprolide, Flutamide (Drogenil), Toremifene (Fareston), and ZOLADEX®.Estrogens stimulate proliferation and differentiation, thereforecompounds that bind to the estrogen receptor are used to block thisactivity. Corticosteroids may inhibit T cell proliferation.

Other chemotherapeutic agents include metal complexes, e.g. cisplatin(cis-DDP), carboplatin, etc.; ureas, e.g. hydroxyurea; and hydrazines,e.g. N-methylhydrazine; epidophyllotoxin; a topoisomerase inhibitor;procarbazine; mitoxantrone; leucovorin; tegafur; etc. Otheranti-proliferative agents of interest include immunosuppressants, e.g.mycophenolic acid, thalidomide, desoxyspergualin, azasporine,leflunomide, mizoribine, azaspirane (SKF 105685); IRESSA® (ZD 1839,4-(3-chloro-4-fluorophenylamino)-7-methoxy-6-(3-(4-morpholinyl)propoxy)quinazoline);etc.

“Taxanes” include paclitaxel, as well as any active taxane derivative orpro-drug. “Paclitaxel” (which should be understood herein to includeanalogues, formulations, and derivatives such as, for example,docetaxel, TAXOL, TAXOTERE (a formulation of docetaxel), 10-desacetylanalogs of paclitaxel and 3′N-desbenzoyl-3′N-t-butoxycarbonyl analogs ofpaclitaxel) may be readily prepared utilizing techniques known to thoseskilled in the art.

Paclitaxel should be understood to refer to not only the commonchemically available form of paclitaxel, but analogs and derivatives(e.g., TAXOTERE™ docetaxel, as noted above) and paclitaxel conjugates(e.g., paclitaxel-PEG, paclitaxel-dextran, or paclitaxel-xylose).

In the treatment of some individuals in accordance with the method ofthe present disclosure, it may be desirable to use a high dose regimenin conjunction with a rescue agent for non-malignant cells. In suchtreatment, any agent capable of rescue of non-malignant cells can beemployed, such as citrovorum factor, folate derivatives, or leucovorin.Such rescue agents are well known to those of ordinary skill in the art.Rescue agents include those which do not interfere with the ability ofthe present inventive compounds to modulate cellular function.

Administration of the Fibrillar Protein

Administration of the fibrillar protein may be achieved through variousmethods to different parts of the body, including intratumoral,intravenous, intradermal, subcutaneous, oral (e.g., inhalation),transdermal (i.e., topical), transmucosal, intraperitoneal,intraarterial, and rectal administration. Other suitable routes includeadministration of the composition orally, bucally, nasally,nasopharyngeally, parenterally, enterically, gastrically, topically,transdermally, subcutaneously, intramuscularly, in tablet, solid,powdered, liquid, aerosol form, intralesional injection into the tumor,intralesional injection adjacent to the tumor, intravenous infusion, andintraarterial infusion. Administration may be done locally orsystemically, with or without added excipients. Administering can alsobe done via slow release mode at or around tumor sites of a subject.

One skilled in the art will appreciate that a variety of suitablemethods of administering a formulation of the present disclosure to asubject or host, e.g., patient, in need thereof, are available, and,although more than one route can be used to administer a particularformulation, a particular route can provide a more immediate and moreeffective reaction than another route.

The phrase “therapeutically effective amount” refers to an amount thatproduces some desired effect at a reasonable benefit/risk ratioapplicable to any medical treatment. The effective amount may varydepending on such factors as the disease or condition being treated, theparticular targeted constructs being administered, the size of thesubject, or the severity of the disease or condition. One of ordinaryskill in the art may empirically determine the effective amount of aparticular compound without necessitating undue experimentation.

According to exemplary implementations, the protein may be administeredas part of a composition, which is described in more detail below. Thecomposition may be in various forms including powders, creams, gels,salves, ointments, solutions, tablets, capsules, sprays, and patches.Vehicles and carriers may be used for delivery of the composition to thepatient. Such carriers include solubilizing agents, diluents, anddispersion media. These carriers are biocompatible, pharmaceuticallyacceptable, and do not alter the treatment characteristics of thefibrillar protein. Excipients, adjuvants and other ingredients may alsobe included in the composition.

Dosage

In the methods, an effective amount of a fibrillar protein isadministered to a subject in need thereof. In particular, fibrillarproteins of specific interest are those that inhibit metastasis of acancer in a host when the fibrillar proteins are administered in aneffective amount. The amount administered varies depending upon the goalof the administration, the health and physical condition of theindividual to be treated, age, the taxonomic group of individual to betreated (e.g., human, non-human primate, primate, etc.), the degree ofresolution desired, the formulation of the fibrillar proteincomposition, the treating clinician's assessment of the medicalsituation, and other relevant factors. It is expected that the amountwill fall in a relatively broad range that can be determined throughroutine trials. For example, the amount of fibrillar protein employed toinhibit cancer metastasis is not more than about the amount that couldotherwise be irreversibly toxic to the subject (i.e., maximum tolerateddose). In other cases the amount is around or even well below the toxicthreshold, but still in an immunoeffective concentration range, or evenas low as threshold dose.

Individual doses are typically not less than an amount required toproduce a measurable effect on the subject, and may be determined basedon the pharmacokinetics and pharmacology for absorption, distribution,metabolism, and excretion (“ADME”) of the fibrillar protein of itsby-products, and thus based on the disposition of the composition withinthe subject. This includes consideration of the route of administrationas well as dosage amount, which can be adjusted for topical (applieddirectly where action is desired for mainly a local effect), enteral(applied via digestive tract for systemic or local effects when retainedin part of the digestive tract), or parenteral (applied by routes otherthan the digestive tract for systemic or local effects) applications.For instance, administration of the fibrillar protein is typically viainjection and often intravenous, intramuscular, intratumoral, or acombination thereof.

The fibrillar protein may be administered by infusion or by localinjection, e.g. by infusion at a rate of about 50 mg/h to about 400mg/h, including about 75 mg/h to about 375 mg/h, about 100 mg/h to about350 mg/h, about 150 mg/h to about 350 mg/h, about 200 mg/h to about 300mg/h, about 225 mg/h to about 275 mg/h. Exemplary rates of infusion canachieve a desired therapeutic dose of, for example, about 0.5 mg/m²/dayto about 10 mg/m²/day, including about 1 mg/m²/day to about 9 mg/m²/day,about 2 mg/m²/day to about 8 mg/m²/day, about 3 mg/m²/day to about 7mg/m²/day, about 4 mg/m²/day to about 6 mg/m²/day, about 4.5 mg/m²/dayto about 5.5 mg/m²/day. Administration (e.g, by infusion) can berepeated over a desired period, e.g., repeated over a period of about 1day to about 5 days or once every several days, for example, about fivedays, over about 1 month, about 2 months, etc. It also can beadministered prior, at the time of, or after other therapeuticinterventions, such as surgical intervention to remove cancerous cells.The fibrillar protein can also be administered as part of a combinationtherapy, in which at least one of an immunotherapy, a cancerchemotherapy or a radiation therapy is administered to the subject (asdescribed in greater detail below).

Disposition of the fibrillar protein and its corresponding biologicalactivity within a subject is typically gauged against the fraction offibrillar protein present at a target of interest. For example, afibrillar protein once administered can accumulate with a glycoconjugateor other biological target that concentrates the material in cancercells and cancerous tissue. Thus dosing regimens in which the fibrillarprotein is administered so as to accumulate in a target of interest overtime can be part of a strategy to allow for lower individual doses. Thiscan also mean that, for example, the dose of fibrillar protein that arecleared more slowly in vivo can be lowered relative to the effectiveconcentration calculated from in vitro assays (e.g., effective amount invitro approximates mM concentration, versus less than mM concentrationsin vivo).

As an example, the effective amount of a dose or dosing regimen can begauged from the IC₅₀ of a given fibrillar protein for inhibiting cellmigration. By “IC₅₀” is intended the concentration of a drug requiredfor 50% inhibition in vitro. Alternatively, the effective amount can begauged from the EC₅₀ of a given fibrillar protein concentration. By“EC₅₀” is intended the plasma concentration required for obtaining 50%of a maximum effect in vivo. In related embodiments, dosage may also bedetermined based on ED₅₀ (effective dosage).

In general, with respect to the fibrillar protein of the presentdisclosure, an effective amount is usually not more than 200× thecalculated IC₅₀. Typically, the amount of an fibrillar protein that isadministered is less than about 200×, less than about 150×, less thenabout 100× and many embodiments less than about 75×, less than about60×, 50×, 45×, 40×, 35×, 30×, 25×, 20×, 15×, 10× and even less thanabout 8× or 2× than the calculated IC₅₀. In one embodiment, theeffective amount is about 1× to 50× of the calculated IC₅₀, andsometimes about 2× to 40×, about 3× to 30× or about 4× to 20× of thecalculated IC₅₀. In other embodiments, the effective amount is the sameas the calculated IC₅₀, and in certain embodiments the effective amountis an amount that is more than the calculated IC₅₀.

An effect amount may not be more than 100× the calculated EC₅₀. Forinstance, the amount of fibrillar protein that is administered is lessthan about 100×, less than about 50×, less than about 40×, 35×, 30×, or25× and many embodiments less than about 20×, less than about 15× andeven less than about 10×, 9×, 9×, 7×, 6×, 5×, 4×, 3×, 2× or 1× than thecalculated EC₅₀. The effective amount may be about 1× to 30× of thecalculated EC₅₀, and sometimes about 1× to 20×, or about 1× to 10× ofthe calculated EC₅₀. The effective amount may also be the same as thecalculated EC₅₀ or more than the calculated EC₅₀. The IC₅₀ can becalculated by inhibiting cell migration/invasion in vitro. The procedurecan be carry out by methods known in the art or as described in theexamples below.

Effective amounts of dose and/or dose regimen can readily be determinedempirically from assays, from safety and escalation and dose rangetrials, individual clinician-patient relationships, as well as in vitroand in vivo assays such as those described herein and illustrated in theExperimental section, below. For example, a concentration used forcarrying out the subject method in mice ranges from about 1 mg/kg toabout 25 mg/kg based on the body weight of the mice. Based on this data,an example of a concentration of the fibrillar protein that can beemployed in human may range about 0.083 mg/kg to about 2.08 mg/kg. Otherdosage may be determined from experiments with animal models usingmethods known in the art (Reagan-Shaw et al. (2007) The FASEB Journal22:659-661).

Pharmaceutical Formulations

Also provided are pharmaceutical compositions containing the fibrillarprotein employed in the methods of treatment described above. The term“fibrillar protein composition” is used herein as a matter ofconvenience to refer generically to compositions comprising a fibrillarprotein of the present disclosure, including conjugated fibrillarprotein, or both. Compositions useful for suppression the growth and/ormetastasis of cancer cells are described below.

The fibrillar protein compositions, e.g., in the form of apharmaceutically acceptable salt, can be formulated for oral, topical orparenteral administration, as described above. In certain embodiments,e.g., where a fibrillar protein is administered as a liquid injectable(such as in those embodiments where they are administered intravenouslyor directly into a tissue), an fibrillar protein formulation is providedas a ready-to-use dosage form, or as a reconstitutable storage-stablepowder or liquid composed of pharmaceutically acceptable carriers andexcipients.

Methods for producing fibrillar protein suitable for administration to asubject (e.g., a human subject) are described below and also in US PatPub No. 2008/0300186, disclosure of which is incorporated by reference.An example method of formulating fibrillar protein can involve apharmaceutical composition containing an effective amount of a fibrillarprotein and a pharmaceutical excipients (e.g., saline). Thepharmaceutical composition may optionally include other additives (e.g.,buffers, stabilizers, preservatives, and the like). An effective amountof fibrillar protein can be an amount effective to provide for adecrease of cancer metastasis (e.g. cancer migration and/or invasion). Atherapeutic goal (e.g., reduction in tumor load and/or confinement ofcancerous growth) can be accomplished by single or multiple doses undervarying dosing regimen.

The concentration of fibrillar protein in the pharmaceuticalformulations can vary from less than about 0.1%, usually at or at leastabout 2% to as much as 20% to 50% or more by weight, and will beselected primarily by fluid volumes, viscosities, etc., in accordancewith the particular mode of administration selected and the patient'sneeds. The resulting compositions may be in the form of a solution,suspension, tablet, pill, capsule, powder, gel, cream, lotion, ointment,aerosol or the like. Actual methods for preparing parenterallyadministrable compositions will be known or apparent to those skilled inthe art and are described in more detail in such publications asRemington's Pharmaceutical Science, 18th ed., Mack Publishing Company,NY (1995).

According to another aspect of this disclosure, fibrillar HSA can beincluded in a pharmaceutical or nutraceutical composition together withadditional active agents, carriers, vehicles, excipients, or auxiliaryagents identifiable by a person skilled in the art upon reading of thepresent disclosure.

The pharmaceutical or nutraceutical compositions preferably comprise atleast one pharmaceutically acceptable carrier. In such pharmaceuticalcompositions, the fibrillar HSA or fibrillar HSA equivalent forms the“active compound,” also referred to as the “active agent.” As usedherein the language “pharmaceutically acceptable carrier” includessolvents, dispersion media, coatings, antibacterial and antifungalagents, isotonic and absorption delaying agents, and the like,compatible with pharmaceutical administration. Supplementary activecompounds can also be incorporated into the compositions. Apharmaceutical composition is formulated to be compatible with itsintended route of administration. Examples of routes of administrationinclude parenteral, e.g., intravenous, intradermal, subcutaneous, oral(e.g., inhalation), transdermal (topical), transmucosal, and rectaladministration. Solutions or suspensions used for parenteral,intradermal, or subcutaneous application can include the followingcomponents: a sterile diluent such as water for injection, salinesolution, fixed oils, polyethylene glycols, glycerine, propylene glycol,or other synthetic solvents; antibacterial agents such as benzyl alcoholor methyl parabens; antioxidants such as ascorbic acid or sodiumbisulfite; chelating agents such as ethylenediaminetetraacetic acid;buffers such as acetates, citrates, or phosphates and agents for theadjustment of tonicity such as sodium chloride or dextrose. pH can beadjusted with acids or bases, such as hydrochloric acid or sodiumhydroxide. The parenteral preparation can be enclosed in ampoules,disposable syringes, or multiple dose vials made of glass or plastic.

Pharmaceutical compositions suitable for an injectable use includesterile aqueous solutions (where water soluble) or dispersions andsterile powders for the extemporaneous preparation of sterile injectablesolutions or dispersion. For intravenous administration, suitablecarriers include physiological saline, bacteriostatic water, Cremophor®EL (BASF, Parsippany, N.J.), or phosphate buffered saline (PBS). In allcases, the composition must be sterile and should be fluid to the extentthat easy syringability exists. It should be stable under the conditionsof manufacture and storage and must be preserved against thecontaminating action of microorganisms such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (for example, glycerol, propylene glycol, andliquid polyetheylene glycol, and the like), and suitable mixturesthereof. The proper fluidity can be maintained, for example, by the useof a coating such as lecithin, by the maintenance of the requiredparticle size in the case of dispersion and by the use of surfactants.Prevention of the action of microorganisms can be achieved by variousantibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, ascorbic acid, thimerosal, and the like.According to embodiments, isotonic agents, for example, sugars,polyalcohols such as manitol, sorbitol, or sodium chloride in thecomposition are added. Prolonged absorption of the injectablecompositions can be brought about by including in the composition anagent which delays absorption, for example, aluminum monostearate andgelatin.

Sterile injectable solutions can be prepared by incorporating the activecompound in the required amount in an appropriate solvent with one or acombination of ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the active compound into a sterile vehicle which containsa basic dispersion medium and the required other ingredients from thoseenumerated above. In the case of sterile powders for the preparation ofsterile injectable solutions, the preparation is prepared by vacuumdrying or freeze-drying, which yields a powder of the active ingredientplus any additional desired ingredient from a previouslysterile-filtered solution thereof.

It is recognized that when administered orally, fibrillar protein shouldbe protected from digestion. This is typically accomplished either bycomplexing the fibrillar protein with a composition to render itresistant to acidic and enzymatic hydrolysis or by packaging in anappropriately resistant carrier such as a liposome. Means of protectinga compound of interest from digestion are well known in the art.

Oral compositions generally include an inert diluent or an ediblecarrier. For the purpose of oral therapeutic administration, the activecompound can be incorporated with excipients and used in the form oftablets, troches, or capsules, e.g., gelatin capsules. Oral compositionscan also be prepared using a fluid carrier for use as a mouthwash.Pharmaceutically compatible binding agents, or adjuvant materials can beincluded as part of the composition. The tablets, pills, capsules,troches and the like can contain any of the following ingredients, orcompounds of a similar nature: a binder such as microcrystallinecellulose, gum tragacanth or gelatin; an excipient such as starch orlactose, a disintegrating agent such as alginic acid, Primogel, or cornstarch; a lubricant such as magnesium stearate or Sterotes; a glidantsuch as colloidal silicon dioxide; a sweetening agent such as sucrose orsaccharin; or a flavoring agent such as peppermint, methyl salicylate,or strawberry, cherry, grape, lemon, or orange flavoring.

For administration by inhalation, the compounds are delivered in theform of an aerosol spray from pressured container or dispenser whichcontains a suitable propellant, e.g., a gas such as carbon dioxide, or anebulizer.

In order to enhance serum half-life, fibrillar protein preparations thatare injected may also be encapsulated, introduced into the lumen ofliposomes, prepared as a colloid, PEGylated (Greenwald et al. (2003)Advanced Drug Delivery Rev. 55:217-250; Pasut et al. (2004) Expert Opin.Ther. Patents 14:859-894) or other conventional techniques may beemployed which provide an extended serum half-life. A variety of methodsare available for preparing liposomes, as described in, e.g., Szoka etal. (1980) Ann. Rev. Biophys. Bioeng. 9:467, U.S. Pat. Nos. 4,235,871,4,501,728 and 4,837,028. The preparations may also be provided incontrolled release or slow-release forms for release and administrationof the fibrillar protein compositions as a mixture or in serial fashion.

According to embodiments, intravitreal injection is accomplished usingPLGA-based microparticles or nanoparticles (liposomes). PEG-basedformulas may also be used. Accordingly, the other methods for injectablepharmaceutical compositions are expressly contemplated for intravitrealinjection.

Systemic administration can also be transmucosal or transdermal. Fortransmucosal or transdermal administration, penetrants appropriate tothe barrier to be permeated are used in the formulation. Such penetrantsare generally known in the art, and include, for example, fortransmucosal administration, detergents, bile salts, and fusidic acidderivatives. Transmucosal administration can be accomplished through theuse of nasal sprays or suppositories. For transdermal administration,the active compounds are formulated into ointments, salves, gels, orcreams as generally known in the art. The compounds can also be preparedin the form of suppositories (e.g., with conventional suppository basessuch as cocoa butter and other glycerides) or retention enemas forrectal delivery.

In addition to the other forms of delivery, the compounds aredeliverable via eye drop or intraocular injection. With respect to eyedrops, the compositions of the present disclosure optionally compriseone or more excipients intended for topical application to the eye ornose. Excipients commonly used in pharmaceutical compositions intendedfor topical application to the eyes, such as solutions or sprays,include, but are not limited to, tonicity agents, preservatives,chelating agents, buffering agents, surfactants and antioxidants.Suitable tonicity-adjusting agents include mannitol, sodium chloride,glycerin, sorbitol and the like. Suitable preservatives includep-hydroxybenzoic acid ester, benzalkonium chloride, benzododeciniumbromide, polyquaternium-1 and the like. Suitable chelating agentsinclude sodium edetate and the like. Suitable buffering agents includephosphates, borates, citrates, acetates and the like. Suitablesurfactants include ionic and nonionic surfactants, though nonionicsurfactants are preferred, such as polysorbates, polyethoxylated castoroil derivatives and oxyethylated tertiary octylphenol formaldehydepolymer (tyloxapol). Suitable antioxidants include sulfites, ascorbates,BHA and BHT. The compositions of the present disclosure optionallycomprise an additional active agent. With the exception of the optionalpreservative ingredient (e.g., polyquaternium-1), the compositions ofthe present disclosure preferably do not contain any polymericingredient other than polyvinylpyrrolidone or polystyrene sulfonic acid.

When the compositions of the present disclosure containpolyvinylpyrrolidone, the polyvinylpyrrolidone ingredient is preferablyselected or processed to minimize peroxide content. Freshly producedbatches of polyvinylpyrrolidone are preferred over aged batches.Additionally, particularly in cases where the composition will containgreater than 0.5% polyvinylpyrrolidone, the polyvinylpyrrolidoneingredient should be thermally treated (i.e., heated to a temperatureabove room temperature) prior to mixing with olopatadine in order toreduce the amount of peroxides in the polyvinylpyrrolidone ingredientand minimize the effect of peroxides on the chemical stability ofolopatadine. While thermally treating an aqueous solution ofpolyvinylpyrrolidone for prolonged periods will substantially reduce theamount of peroxides, it can lead to discoloration (yellow toyellowish-brown) of the polyvinylpyrrolidone solution. In order tosubstantially reduce or eliminate peroxides without discoloring thepolyvinylpyrrolidone solution, the pH of the aqueous solution ofpolyvinylpyrrolidone should be adjusted to pH 11-13 before it issubjected to heat. Much shorter heating times are needed to achievesignificant reductions in peroxide levels if the pH of thepolyvinylpyrrolidone solution is elevated.

One suitable method of thermally treating the polyvinylpyrrolidoneingredient is as follows. First, dissolve the polyvinylpyrrolidoneingredient in purified water to make a 4-6% solution, then raise the pHof the solution to pH 11-13, (an effective range of pH is 11-11.5), thenheat to a temperature in the range of 60-121° C., preferably 65-80° C.and most preferably 70-75° C. The elevated temperature should bemaintained for approximately 30-120 minutes (preferably 30 minutes).After the heated solution cools to room temperature, add HCl to adjustthe pH to 3.5-8, depending upon the target pH for the olopatadinecomposition.

Particularly for compositions intended to be administered as eye drops,the compositions preferably contain a tonicity-adjusting agent in anamount sufficient to cause the final composition to have anophthalmically acceptable osmolality (generally 150-450 mOsm, preferably250-350 mOsm). The ophthalmic compositions of the present disclosurepreferably have a pH of 4-8, preferably a pH of 6.5-7.5, and mostpreferably a pH of 6.8-7.2.

The eye-drop compositions of the present disclosure are preferablypackaged in opaque plastic containers. A preferred container for anophthalmic product is a low-density polyethylene container that has beensterilized using ethylene oxide instead of gamma-irradiation.

With respect to ophthalmic injectables, the pharmaceutical compositionsof this disclosure are administered to the area in need of treatment bysubconjunctival administration. One preferred method of subconjunctivaladministration to the eye is by injectable formulations comprising thepharmaceutical compositions disclosed herein. Another preferred methodof subconjunctival administration is by implantations comprising slowreleasing compositions.

Compositions that are delivered subconjunctivally comprise, according toembodiments, an ophthalmic depot formulation comprising an active agentfor subconjunctival administration. According to embodiments, theophthalmic depot formulation comprises microparticles of essentiallypure active agent. The microparticles comprising can be embedded in abiocompatible pharmaceutically acceptable polymer or a lipidencapsulating agent. The depot formulations may be adapted to releaseall of substantially all the active material over an extended period oftime. The polymer or lipid matrix, if present, may be adapted to degradesufficiently to be transported from the site of administration afterrelease of all or substantially all the active agent. The depotformulation can be liquid formulation, comprising a pharmaceuticalacceptable polymer and a dissolved or dispersed active agent. Uponinjection, the polymer forms a depot at the injections site, e.g., bygelifying or precipitating.

Solid articles suitable for implantation in the eye can also be designedin such a fashion to comprise polymers and can be bioerodible ornon-bioerodible. Bioerodible polymers that can be used in preparation ofocular implants carrying the compositions of the present disclosureinclude without restriction aliphatic polyesters such as polymers andcopolymers of poly(glycolide), poly(lactide), poly(c-caprolactone),poly(hydroxybutyrate) and poly(hydroxyvalerate), polyamino acids,polyorthoesters, polyanhydrides, aliphatic polycarbonates and polyetherlactones. Illustrative of suitable non-bioerodible polymers are siliconeelastomers.

According to embodiments, the active compounds are prepared withcarriers that will protect the compound against rapid elimination fromthe body, such as a controlled release formulation, including implantsand microencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid.Methods for preparation of such formulations will be apparent to thoseskilled in the art. The materials can also be obtained commercially fromAlza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions(including liposomes targeted to infected cells with monoclonalantibodies to cell-specific antigens) can also be used aspharmaceutically acceptable carriers.

The fibrillar protein composition can be administered as a singlepharmaceutical formulation. It may also be administered with aneffective amount of another agent that includes other suitable compoundsand carriers, and also may be used in combination with other activeagents. The present disclosure, therefore, also includes pharmaceuticalcompositions comprising pharmaceutically acceptable excipients.

The pharmaceutically acceptable excipients include, for example, anysuitable vehicles, adjuvants, carriers or diluents, and are readilyavailable to the public. The pharmaceutical compositions of the presentdisclosure may further contain other active agents as are well known inthe art. Pharmaceutically acceptable excipients are also well-known tothose who are skilled in the art, and are readily available.

For example, the fibrillar protein compositions can be admixed withconventional pharmaceutically acceptable carriers and excipients (i.e.,vehicles) and used in the form of aqueous solutions, tablets, capsules,elixirs, suspensions, syrups, wafers, patches and the like, but usuallythe fibrillar protein will be provided as an injectable. Thepharmaceutical compositions may contain common carriers and excipients,such as corn starch or gelatin, lactose, dextrose, sucrose,microcrystalline cellulose, kaolin, mannitol, dicalcium phosphate,sodium chloride, and alginic acid. Disintegrators commonly used informulations include croscarmellose, microcrystalline cellulose, cornstarch, sodium starch glycolate and alginic acid. Preservatives and thelike may also be included. Each of these components is well-known in theart. Such pharmaceutical compositions contain, in certain embodiments,from about 0.1 to about 90% by weight of the active compound, and moregenerally from about 1 to about 30% by weight of the active compound.See, e.g., U.S. Pat. No. 5,985,310, the disclosure of which is hereinincorporated by reference

The fibrillar protein compositions can be provided in a pharmaceuticallyacceptable excipient, which can be a solution such as an aqueoussolution, often a saline solution, or they can be provided in powderform. The fibrillar protein compositions may contain other components,such as pharmaceutical grades of mannitol, lactose, starch, magnesiumstearate, sodium saccharin, talcum, cellulose, glucose, sucrose,magnesium, carbonate, and the like. The compositions may containpharmaceutically acceptable auxiliary substances as required toapproximate physiological conditions such as pH adjusting and bufferingagents, toxicity adjusting agents and the like, for example, sodiumacetate, sodium chloride, potassium chloride, calcium chloride, sodiumlactate and the like.

The choice of excipient will be determined in part by the particularcompound, as well as by the particular method used to administer thecomposition. Accordingly, there is a wide variety of suitableformulations of the pharmaceutical composition of the presentdisclosure. The following methods and excipients are merely exemplaryand are in no way limiting.

A liquid composition will generally be composed of a suspension orsolution of the compound or pharmaceutically acceptable salt in asuitable liquid carrier(s), for example, ethanol, glycerine, sorbitol,non-aqueous solvent such as polyethylene glycol, oils or water, with asuspending agent, preservative, surfactant, wetting agent, flavoring orcoloring agent. Alternatively, a liquid formulation can be prepared froma reconstitutable powder.

Formulations suitable for parenteral administration include aqueous andnon-aqueous, isotonic sterile injection solutions, which can containanti-oxidants, buffers, bacteriostats, and solutes that render theformulation isotonic with the blood of the intended recipient, andaqueous and non-aqueous sterile suspensions that can include suspendingagents, solubilizers, thickening agents, stabilizers, and preservatives.The formulations can be presented in unit-dose or multi-dose sealedcontainers, such as ampules and vials, and can be stored in afreeze-dried (lyophilized) condition requiring only the addition of thesterile liquid excipient, for example, water, for injections,immediately prior to use. Extemporaneous injection solutions andsuspensions can be prepared from sterile powders, granules, and tabletsof the kind previously described.

The fibrillar proteins of the present disclosure and theirpharmaceutically acceptable salts that are active when givenparenterally can be formulated for intramuscular, intrathecal, orintravenous administration. A typical composition for intramuscular orintrathecal administration will be of a suspension or solution of activeingredient in an oil, for example, arachis oil or sesame oil. A typicalcomposition for intravenous or intrathecal administration will be asterile isotonic aqueous solution containing, for example, activeingredient and dextrose or sodium chloride, or a mixture of dextrose andsodium chloride. Other examples are lactated Ringer's injection,lactated Ringer's plus dextrose injection, Normosol-M and dextrose,Isolyte E, acylated Ringer's injection, and the like. Optionally, aco-solvent, for example, polyethylene glycol, a chelating agent, forexample, ethylenediamine tetracetic acid, and an anti-oxidant, forexample, sodium metabisulphite may be included in the formulation.Alternatively, the solution can be freeze dried and then reconstitutedwith a suitable solvent just prior to administration.

The fibrillar proteins of the present disclosure and theirpharmaceutically acceptable salts which are active on rectaladministration can be formulated as suppositories. A typical suppositoryformulation will generally consist of active ingredient with a bindingand/or lubricating agent such as a gelatin or cocoa butter or other lowmelting vegetable or synthetic wax or fat.

The fibrillar proteins of the present disclosure and theirpharmaceutically acceptable salts which are active on topicaladministration can be formulated as transdermal compositions ortransdermal delivery devices (“patches”). Such compositions include, forexample, a backing, active compound reservoir, a control membrane, linerand contact adhesive. Such transdermal patches may be used to providecontinuous or discontinuous infusion of the compounds of the presentdisclosure in controlled amounts. The construction and use oftransdermal patches for the delivery of pharmaceutical agents is wellknown in the art. See, e.g., U.S. Pat. No. 5,023,252, hereinincorporated by reference in its entirety. Such patches may beconstructed for continuous, pulsatile, or on demand delivery ofpharmaceutical agents.

The formulations of the present disclosure can be made into aerosolformulations to be administered via inhalation. These aerosolformulations can be placed into pressurized acceptable propellants, suchas dichlorodifluoromethane, propane, nitrogen, and the like. They mayalso be formulated as pharmaceuticals for non-pressured preparationssuch as for use in a nebulizer or an atomizer.

Formulations suitable for topical administration may be presented ascreams, gels, pastes, or foams, containing, in addition to the activeingredient, such carriers as are known in the art to be appropriate.

Suppository formulations are also provided by mixing with a variety ofbases such as emulsifying bases or water-soluble bases. Formulationssuitable for vaginal administration may be presented as pessaries,tampons, creams, gels, pastes, foams.

Unit dosage forms for oral or rectal administration such as syrups,elixirs, and suspensions may be provided wherein each dosage unit, forexample, teaspoonful, tablespoonful, tablet or suppository, contains apredetermined amount of the composition containing one or more fibrillarprotein. Similarly, unit dosage forms for injection or intravenousadministration may comprise the fibrillar protein (s) in a compositionas a solution in sterile water, normal saline or anotherpharmaceutically acceptable carrier.

The term “unit dosage form,” as used herein, refers to physicallydiscrete units suitable as unitary dosages for human and animalsubjects, each unit containing a predetermined quantity of compounds ofthe present disclosure calculated in an amount sufficient to produce thedesired effect in association with a pharmaceutically acceptablediluent, carrier or vehicle. The specifications for the novel unitdosage forms of the present disclosure depend on the particular compoundemployed and the effect to be achieved, and the pharmacodynamicsassociated with each compound in the host.

Those of skill in the art will readily appreciate that dose levels canvary as a function of the specific fibrillar protein, the nature of thedelivery vehicle, and the like. Suitable dosages for a given compoundare readily determinable by those of skill in the art by a variety ofmeans.

Toxicity and therapeutic efficacy of such compounds can be determined bystandard pharmaceutical procedures in cell cultures or experimentalanimals, e.g., for determining the LD₅₀ (the dose lethal to 50% of thepopulation) and the ED₅₀ (the dose therapeutically effective in 50% ofthe population). The dose ratio between toxic and therapeutic effects isthe therapeutic index and it can be expressed as the ratio LD₅₀/ED₅₀.Compounds which exhibit high therapeutic indices are preferred. Whilecompounds that exhibit toxic side effects can be used, care should betaken to design a delivery system that targets such compounds to thesite of affected tissue in order to minimize potential damage touninfected cells and, thereby, reduce side effects.

The data obtained from the cell culture assays and animal studies can beused in formulating a range of dosage for use in humans. The dosage ofsuch compounds lies preferably within a range of circulatingconcentrations that include the ED₅₀ with little or no toxicity. Thedosage can vary within this range depending upon the dosage formemployed and the route of administration utilized. For any compound usedin the method of the disclosure, the therapeutically effective dose canbe estimated initially from cell culture assays. A dose can beformulated in animal models to achieve a circulating plasmaconcentration range that includes the IC₅₀ (i.e., the concentration ofthe test compound which achieves a half-maximal inhibition of symptoms)as determined in cell culture. Such information can be used to moreaccurately determine useful doses in humans. Levels in plasma can bemeasured, for example, by high performance liquid chromatography.

The term “effective amount” refers to an amount of a biologically activemolecule or conjugate or derivative thereof sufficient to exhibit adetectable therapeutic effect without undue adverse side effects (suchas toxicity, irritation and allergic response) commensurate with areasonable benefit/risk ratio when used in the manner of the invention.The therapeutic effect may include, for example but not by way oflimitation, being substantially cytotoxic to cancer cells, but lesscytotoxic to natural cells. The effective amount for a subject willdepend upon the type of subject, the subject's size and health, thenature and severity of the condition to be treated, the method ofadministration, the duration of treatment, the nature of concurrenttherapy (if any), the specific formulations employed, and the like.Thus, it is not possible to specify an exact effective amount inadvance. However, the effective amount for a given situation can bedetermined by one of ordinary skill in the art using routineexperimentation based on the information provided herein.

Methods of Making Fibrillar Protein

According to the present disclosure, a method is disclosed for treatingcancer metastasis by administering a fibrillar protein. The fibrillarprotein can be naturally-occurring and as such, can isolated usingmethods known in the art for use in the method of treatment describedabove. The fibrillar protein can also be made artificially by any methodknown in the art, e.g. by changing a globular protein structure into afibrillar protein structure. In one example, fibrillization of proteinscan be induced by a process without the assistance of fibril seed, asdisclosed in US Pat Pub No. 2008/0800186, disclosure of which isincorporated herein by reference. This process has advantages whichinclude ease of control, homogeneity of production, and feasibility ofscaling up. Moreover, fibrillization of proteins can be induced by thisprocess without the assistance of fibril seed. Even a tiny amount ofprotein would be applicable to this process. As used herein, “protein”includes one or more proteins, protein fragments, polypeptides, orpeptides. Proteins include both synthetic and naturally occurringproteins.

In accordance with the method disclosed previously in US Pat Pub No.2008/0800186, even a tiny amount of protein would be applicable to thisprocess. As used herein, “protein” includes one or more proteins,protein fragments, polypeptides or peptides. Proteins include bothsynthetic and naturally occurring proteins. The method can be used toconvert native proteins, regardless of their sequence, into fibrillarform in a simple and rapid manner. The method comprises the steps ofdissolving a globular protein in a solution that contains detergents andapplying the solution to a molecular sizing column that can separateproteins of 70 kDa molecular weight or larger, and eluting the proteinwith a solution containing detergent. In an exemplary implementation,the method comprises the steps of providing a globular protein, forminga solution containing the globular protein, adding a detergent to thesolution containing the globular protein, and applying the solution to amolecular sizing column with a pore size that can separate proteins of70 kDa molecular weight and above, optionally in the presence of lowconcentration of detergent.

Globular proteins, also known as spheroproteins, are one of two maintertiary structure classes of proteins. Globular proteins are generallysoluble and form spheriodal molecules in water. They have a complexsecondary structure comprising a mixture of secondary structure motifs,such as α-helices, β-sheets, and loop structures. The other maintertiary structure class of proteins are fibrillar proteins, or fibrousproteins. Fibrillar proteins are generally insoluble and have anelongated shape. They have a simpler secondary structure and are oftenbased on only one type of secondary structure motif. In examples ofimplementations, the globular protein is an albumin, for example humanserum albumin, fibronectin, etc. Recombinant unfolded proteins extractedfrom the inclusion bodies of E. coli with 8M urea can also be used, forexample recombinant caspid protein VP1 of the foot-and-mouth-diseasevirus (rVP1), recombinant caspid protein VP2 of thefoot-and-mouth-disease virus (rVP2), recombinant caspid protein VP3 ofthe foot-and-mouth-disease virus (rVP3), or precursor protein P1 of VP1,VP2, VP3, and VP4. The protein may also be a chimeric protein comprisingparts from VP1, VP2, VP3, and/or VP4, for example VP42, which comprisesparts of both VP2 and VP4. Other globular proteins (e.g. bovine serumalbumin) may also be used, including both naturally-occurring proteinsand synthetic oligopeptides.

Surfactants, also referred to herein as detergents, are substances thatlower the surface tension of water and increase the solubility oforganic compounds. Detergents may be ionic, which includes cationic,anionic, and zwitterionic detergents, as well as non-ionic. Detergentsplay a role in disrupting non-covalent bonds in proteins, therebydenaturing the proteins such that they lose their native shape orconformation. In exemplary implementations, the detergent used is sodiumdodecyl sulfate (SDS), obtained from Sigma. In other exemplaryimplementations, the detergent used is Zwittergent 3-14, obtained fromCalbiochem.

Amyloids are fibrous cross-β protein aggregates. Numerous proteins arecapable of converting to amyloid-like fibrils with characteristics thatinclude fibrillar morphology, protofilament substructure, cross-βdiffraction pattern, an increase in β-structure, Congo red binding, andThT binding. In exemplary implementations, the globular protein isconverted to form amyloid-like fibrils, which allows for the convertedprotein to be identified by its amyloid-like properties.

The protein for treating the cancer may be selected based on theseverity of the disease and the desired cytotoxicity to the cancercells. In exemplary implementations, for greater cytotoxicity to thecancer cells is selected. The fibrillar proteins may include fibrillarproteins derived from the same protein types or more than one, more thantwo, more than three or more types. For example, the methods oftreatment described above can administer rVP1 fibrillar proteins andfibrillar BSA together in one dosage or separately.

According to implementations, chromatography may be used in the processto convert the globular protein structure into a fibrillar proteinstructure and separate them. Generally, chromatography is accomplishedusing columns, though other methods such as those used for thin-layerchromatography may also be possible. Chromatography techniques includesize exclusion, affinity, and ion-exchange. Though a batch-typeproduction of fibrillar proteins is possible, utilizing a column processallows globular proteins to be converted into a fibrillar form in arapid, steady, efficient, and continuous manner. Scaling-up this processis also possible with the usage of columns.

According to exemplary implementations, size exclusion chromatographywith bead pore sizes of at least about 70 kDa is used. The bead poresize used may vary depending on various characteristics of the globularprotein, for example its size. The pore size plays a role in allowingproteins to enter the bead matrix, thus leading to mechanical forceswhich contribute to protein unfolding/folding and enhance fibrillogenicensemble. In exemplary implementations, the molecular sizing column usedis a Superdex 200. In other exemplary implementations, the molecularsizing column used is a HW55S.

For column chromatography, a buffer solution containing lowconcentration(s) of detergent may be used to elute the column. Inexemplary implementations, the molecular sizing column is eluted with abuffer solution containing 25 mM Tris-HCl (pH 8.0), 1 mM EDTA, 0.1 MNaCl, and 0.05% SDS. In other exemplary implementations, the molecularsizing column is eluted with a buffer solution containing 25 mMTris-HCl, pH 8.0, 1 mM EDTA, 0.1 M NaCl, and 0.05% Zwittergent 3-14. Theeluant may be collected as fractions and the fractions containing thefibrillar protein subsequently pooled together. The pooled fraction maythen be further filtered to purify and isolate the fibrillar protein,for example dialyzing against PBS to remove SDS or Zwittergent 3-14.

According to implementations, human serum albumin (HSA) can be made intofibrillar human serum albumin by the processes disclosed herein forcreating fibrillar proteins. According to implementations, human serumalbumin has been confirmed convert to fibrillar form by the processesdisclosed herein. With respect to creating fibrillar proteins, U.S. Pat.No. 7,488,800 is incorporated by reference.

The fibrillar HSA (F-HSA) was unexpectedly found to be at least aspotent as recombinant capsid protein of foot and mouth disease virus(rVP1) in causing apoptosis in a variety of cancer cells. Among theadvantages of using F-HSA instead of rVP1 as a cancer therapeutic isthat HSA is a human endogenous protein. Thus, HSA or its derivativeswith similar sequence and composition would be less likely than foreignproteins such as rVP1 to induce immunogenicity and neutralizingantibodies during clinical applications.

According to implementations, F-HSA was generated by dissolving HSA in a1% SDS solution, passing through a Superdex-200 gel filtration columnand eluting with a buffer solution containing 25 mM Tris-HCl (pH 8.0), 1mM EDTA, 0.1 M NaCl, and 0.05% SDS (FIG. 1). After dialysis against PBSto remove the SDS, it was found that unlike HSA, the eluted F-HSA fromthe Superdex-200 column exhibited enhanced fluorescence level ofamyloid-specific dye ThT in a dose-dependent manner.

It was then determined that F-HSA induced cytotoxicity in cancer cells.As fibrillar serum albumin bound to receptors such as integrins on thecell surface while globular serum albumin could not, it is believed thatthe change of structure of serum albumin from globular to fibrillar formhas enabled the proteins to selectively target cancer cells thatexpressed more integrin α5β1 than normal cells. F-HSA inhibited breastcancer cell growth dose dependently including TS/A (murine mammaryadenocarcinoma) and MDA-MB-231 (human mammary adenocarcinoma) cells withIC₅₀ of 0.15 (FIG. 13) and 0.48 μM (FIG. 14), respectively. F-HSAinhibited ovarian cancer cell SKOV3 growth with IC₅₀ of 0.6 μM (FIG. 15)and cervical cancer cell CaSki growth with IC₅₀ of 1.1 μM (FIG. 16).F-HSA also induced cytotoxicity in prostate cancer cells PC-3 and 22Rv1with IC₅₀ of 0.35 (FIG. 17) and 0.2 μM (FIG. 18), respectively. Inaddition, F-HSA induces cytotoxicity in a number lung cancer cell lines(FIG. 19).

According to implementations, therefore, a method for treating cancer isdisclosed. The method comprises the steps of providing HSA, changing theHSA into a fibrillar structure, and administering a therapeuticallyeffective amount of the F-HSA to a patient in need thereof. Conversionof the HSA into fibrillar form increases its cytotoxic effects on targetcells.

In exemplary implementations, the cancer is a kidney, breast, lung,prostate, liver, cervical, or ovarian cancer. In exemplaryimplementations, the fibrillar HSA plays a role in inducing cancer cellapoptosis by modulating the Akt signaling pathway. In some instances,the fibrillar HSA modulates integrin α5β1 or αvβ3 which leads to thedeactivation of Akt. In other instances, fibrillar HSA binds to integrinand causes cellular apoptosis mainly through theintegrin/FAK/Akt/GSK-3β/caspase-3 pathway.

The fibrillar HSA protein, derivate, ortholog, or other protein havingsubstantial identity to HSA for treating the cancer may be selectedbased on the severity of the disease and the desired cytotoxicity to thecancer cells. In exemplary implementations, for greater cytotoxicity tothe cancer cells, a protein with an RGD motif or greater molecularweight is selected. RGD motif is a ligand for integrins. It has beenshown that fibrillar proteins induced cell death via modulatingintegrin/Akt signaling pathway. It has been found that fibrillarproteins with RGD motifs, like rVP1-5200 and FN-5200, were morecytotoxic than those without RGD motifs such as BSA-5200 and rVP3-5200.

“Variant” refers to a polynucleotide or polypeptide that differs from areference polynucleotide or polypeptide, but retains essentialproperties. A typical variant of a polynucleotide differs in nucleotidesequence from another, reference polynucleotide. Changes in thenucleotide sequence of the variant may or may not alter the amino acidsequence of a polypeptide encoded by the reference polynucleotide.Nucleotide changes may result in amino acid substitutions, additions,deletions, fusions, and truncations in the polypeptide encoded by thereference sequence, as discussed herein.

A typical variant of a polypeptide differs in amino acid sequence fromanother, reference polypeptide. Generally, differences are limited sothat the sequences of the reference polypeptide and the variant areclosely similar overall and, in many regions, identical. A variant andreference polypeptide may differ in amino acid sequence by one or moresubstitutions, additions, and deletions in any combination. Asubstituted or inserted amino acid residue may or may not be one encodedby the genetic code. A variant of a polynucleotide or polypeptide may bea naturally occurring such as an allelic variant, or it may be a variantthat is not known to occur naturally. Non-naturally occurring variantsof polynucleotides and polypeptides may be made by mutagenesistechniques or by direct synthesis.

An “ortholog” denotes a polypeptide or polynucleotide obtained fromanother species that is the functional counterpart of a polypeptide orpolynucleotide from a different species. Sequence differences amongorthologs are the result of speciation.

As used herein, the twenty conventional amino acids and theirabbreviations follow conventional usage. See Immunology—A Synthesis (2ndEdition, E. S. Golub and D. R. Gren, Eds., Sinauer Associates,Sunderland, Mass. (1991)), which is incorporated herein by reference.Stereoisomers (e.g., D-amino acids) of the twenty conventional aminoacids, unnatural amino acids such as α-,α-disubstituted amino acids,N-alkyl amino acids, lactic acid, and other unconventional amino acidsmay also be suitable components for polypeptides of the presentinvention. Examples of unconventional amino acids include:4-hydroxyproline, γ-carboxyglutamate, ε-N,N,N-trimethyllysine,ε-N-acetyllysine, O-phosphoserine, N-acetylserine, N-formylmethionine,3-methylhistidine, 5-hydroxylysine, σ-N-methylarginine, and othersimilar amino acids and imino acids (e.g., 4-hydroxyproline). In thepolypeptide notation used herein, the lefthand direction is the aminoterminal direction and the righthand direction is the carboxy-terminaldirection, in accordance with standard usage and convention.

As applied to polypeptides, the term “substantial identity” means thattwo peptide sequences, when optimally aligned, such as by the programsGAP or BESTFIT using default gap weights, share at least 80 percentsequence identity, preferably at least 90 percent sequence identity,more preferably at least 95 percent sequence identity, and mostpreferably at least 99 percent sequence identity. Preferably, residuepositions which are not identical differ by conservative amino acidsubstitutions. Conservative amino acid substitutions refer to theinterchangeability of residues having similar side chains. For example,a group of amino acids having aliphatic side chains is glycine, alanine,valine, leucine, and isoleucine; a group of amino acids havingaliphatic-hydroxyl side chains is serine and threonine; a group of aminoacids having amide-containing side chains is asparagine and glutamine; agroup of amino acids having aromatic side chains is phenylalanine,tyrosine, and tryptophan; a group of amino acids having basic sidechains is lysine, arginine, and histidine; and a group of amino acidshaving sulfur-containing side chains is cysteine and methionine.Preferred conservative amino acids substitution groups are:valine-leucine-isoleuci-ne, phenylalanine-tyrosine, lysine-arginine,alanine-valine, glutamic-aspartic, and asparagine-glutamine.

As discussed herein, minor variations in the amino acid sequences ofcompositions having cytotoxic activities are contemplated as beingencompassed by the present invention, providing that the variations inthe amino acid of HSA sequence maintain at least 75%, at least 80%, atleast 90%, at least 95%, or at least 99% identity. In particular,conservative amino acid replacements are contemplated. Conservativereplacements are those that take place within a family of amino acidsthat are related in their side chains. Genetically encoded amino acidsare generally divided into families: (1) acidic=aspartate, glutamate;(2) basic=lysine, arginine, histidine; (3) nonpolar=alanine, valine,leucine, isoleucine, proline, phenylalanine, methionine, tryptophan; and(4) uncharged polar=glycine, asparagine, glutamine, cysteine, serine,threonine, tyrosine. More preferred families are: serine and threonineare aliphatic-hydroxy family; asparagine and glutamine are anamide-containing family; alanine, valine, leucine and isoleucine are analiphatic family; and phenylalanine, tryptophan, and tyrosine are anaromatic family. For example, it is reasonable to expect that anisolated replacement of a leucine with an isoleucine or valine, anaspartate with a glutamate, a threonine with a serine, or a similarreplacement of an amino acid with a structurally related amino acid willnot have a major effect on the binding or properties of the resultingmolecule, especially if the replacement does not involve an amino acidwithin a framework site. Whether an amino acid change results in afunctional peptide can readily be determined by assaying the specificactivity of the polypeptide derivative. Fragments or analogs of proteinsor peptides of the present invention can be readily prepared by those ofordinary skill in the art. Preferred amino- and carboxy-termini offragments or analogs occur near boundaries of functional domains.Structural and functional domains can be identified by comparison of thenucleotide or amino acid sequence data to public or proprietary sequencedatabases. Preferably, computerized comparison methods are used toidentify sequence motifs or predicted protein conformation domains thatoccur in other proteins of known structure or function. Methods toidentify protein sequences that fold into a known three-dimensionalstructure are known. Bowie et al. Science 253:164 (1991). Thus, theforegoing examples demonstrate that those of skill in the art canrecognize sequence motifs and structural conformations that may be usedto define structural and functional domains in accordance with theinvention.

Effective amino acid substitutions are those which: (1) reducesusceptibility to proteolysis, (2) reduce susceptibility to oxidation,(3) alter binding affinity for forming protein complexes, (4) alterbinding affinities, and (5) confer or modify other physicochemical orfunctional properties of such analogs. Analogs can include variousmutations of a sequence other than the naturally-occurring peptidesequence. For example, single or multiple amino acid substitutions(preferably conservative amino acid substitutions) may be made in thenaturally-occurring sequence (preferably in the portion of thepolypeptide outside the domain(s) forming intermolecular contacts. Aconservative amino acid substitution should not substantially change thestructural characteristics of the parent sequence (e.g., a replacementamino acid should not tend to break a helix that occurs in the parentsequence, or disrupt other types of secondary structure thatcharacterizes the parent sequence). Examples of art-recognizedpolypeptide secondary and tertiary structures are described in Proteins,Structures and Molecular Principles (Creighton, Ed., W. H. Freeman andCompany, New York (1984)); Introduction to Protein Structure (C. Brandenand J. Tooze, eds., Garland Publishing, New York, N.Y. (1991)); andThornton et at. Nature 354:105 (1991), which are each incorporatedherein by reference.

The term “polypeptide fragment” as used herein refers to a polypeptidethat has an amino-terminal or carboxy-terminal deletion, but where theremaining amino acid sequence is identical to the correspondingpositions in the naturally-occurring sequence deduced, for example, froma full-length cDNA sequence. Fragments typically are at least 5, 6, 8 or10 amino acids long, preferably at least 14 amino acids long, morepreferably at least 20 amino acids long, usually at least 50 amino acidslong, and even more preferably at least 70 amino acids long.

Generally, the method of making fibrillar proteins involves dissolvingproteins in a SDS or other suitable detergents solution; applying thedissolved proteins through a gel filtration column with a pore size thatcan separate proteins of 70 kDa molecular weight and above; eluting theprotein from the column; and dialyzing the eluate against bufferedsaline to remove the SDS or detergents.

In the first step, the globular protein is generally dissolved intosolution form. In an example, the globular protein is dissolved in PBSwith surfactants. Surfactants, also referred to herein as detergents,are substances that lower the surface tension of water and increase thesolubility of organic compounds. Detergents may be ionic, which includescationic, anionic, and zwitterionic detergents, as well as non-ionic.Detergents play a role in disrupting non-covalent bonds in proteins,thereby denaturing the proteins such that they lose their native shapeor conformation. In exemplary implementations, the detergent used issodium dodecyl sulfate (SDS), obtained from Sigma. In other exemplaryimplementations, the detergent used is Zwittergent 3-14, obtained fromCalbiochem.

In some aspects of the method, size exclusion chromatography with beadpore sizes of at least about 70 kDa is used. The bead pore size used mayvary depending on various characteristics of the globular protein, forexample its size. The pore size plays a role in allowing proteins toenter the bead matrix, thus leading to mechanical forces whichcontribute to protein unfolding/folding and enhance fibrillogenicensemble. In exemplary implementations, the molecular sizing column usedis a Superdex 200. In other exemplary implementations, the molecularsizing column used is a HW55S. Other details of the method to producefibrillar proteins may be found in US Pat Pub No. 2008/0800186,disclosure of which is incorporated herein by reference.

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the subject invention, and are not intended to limit thescope of what is regarded as the invention.

EXPERIMENTAL Example 1 Methods

Cell Lines and Culture.

SK-OV-3 cells (human ovarian carcinoma cell line; ATCC HTB-77) andSK-OV-3ip.1 cells, and CaSki cells (human cervical carcinoma cell line;ATCC CRL-1550) were maintained at 37° C. in McCoy's 5A and RPMI-1640medium, respectively. MDA-MB-231 cells (human mammary adenocarcinomacell line; ATCC HTB-26) and TS/A cells (murine mammary adenocarcinomacell line) were maintained at 37° C. in Dulbecco's modified Eagle'smedium (DMEM)/F12 medium and DMEM, respectively. PC-3 cells (humanprostate adenocarcinoma cell line; ATCC CRL-1435) and 22Rv1 cells (humanprostate carcinoma cell line; ATCC CRL-2505) were maintained at 37° C.in RPMI-1640 medium. All medium were supplemented with 10% fetal bovineserum (FBS), 2 mM L-glutamine, 100 units/ml penicillin, and 100 μg/mlstreptomycin.

Cytotoxicity Assay.

Cell survival was determined by MTT assay or WST-1 assay. In brief,1×10⁵ cells/well of tumor cells were seeded in 96-well plates inserum-free medium and incubated for 1 h. Treatment of cells with aseries of concentrations of rVP1, F-BSA, or F-HSA was carried out inserum-free medium for 24 h at 37° C. After treatment, MTT solution wasadded to each well (0.5 mg/ml), followed by 4 h incubation. The viablecell number is directly proportional to the production of formazan,which, following solubilization with isopropanol, can be measuredspectrophotometrically at 570 nm by an ELISA plate reader. Dataexpressed as a percentage of untreatment condition and presented as themean±S.D.

WST-1 assay was carried out according to the manufacturer's instructions(Roche, Mannheim, Germany). In brief, 2×10⁴ cells were added to 100 μlmedia per well on a 96 well plate and incubated at 37° C. in 5% CO₂overnight in a humidified incubator. The cells attached to the wellswere incubated in serum-free medium and treated with serial dilutions ofrVP1. After incubation at 37° C. in 5% CO₂ for 16 h to allow the drug totake effect, 10 μl WST-1 reagent was added to each well, and the platewas then mixed on a shaking table at 150 rpm for 1 min. After incubationat 37° C. in 5% CO₂ for another 2 h to allow the WST-1 reagent to bemetabolized, the proportion of surviving cells were determined byoptical density (450 nm test wavelength, 690 nm reference wavelength).The percentage of surviving cells was calculated as(O.D._(treatment)/O.D._(control))×100% while the percentage of growthinhibition was calculated as [1−(O.D._(treatment)/O.D._(control))]×100%.IC₅₀ is the concentration at which the reagent yields 50% inhibition ofthe cellular viability.

Cell Migration and Invasion Assay.

Cell migration and invasion assays were performed following Boydenchamber migration and invasion assay (Corning). The 8-μm pore membranesof the upper chambers were coated with 20 μg/ml fibronectin for cellmigration assay or 40 μg/ml Matrigel for cell invasion assay and placedin a well with 1 ml of PBS and incubated for 2 hours at 37° C. Cancercells (1×10⁵) were re-suspended in serum-free medium and plated onto theupper chamber for 1 h. A various concentration of fibrillar proteinssuch as rVP1 were added into the upper chamber then the culture medium(10% FBS medium) was added to the lower chamber. Cells were incubatedfor 24 hours at 37° C. At the end point of incubation, cells on theupper side of the membrane were removed by wiping it with a cotton swab,and cells that had migrated onto the lower membrane surface weredissociated by using cell dissociation solution (Sigma) and counted byflow cytometer (BD company).

Establishment of Metastatic Implantation in a Murine Model of TS/AMammary Adenocarcinoma in BALB/c Mice.

Murine TS/A mammary adenocarcinoma cells were cultured in DMEMsupplemented with 10% FBS, 2 mM L-glutamine, 100 U/ml penicillin and 100μg/ml streptomycin in a humidified atmosphere of 95% air and 5% CO₂ at37° C. After harvest, TS/A cells (3×10⁵/100 μl PBS/mouse) wereintravenously injected into the lateral tail vein of nine mice pergroup. Control medium or fibrillar proteins such as F-HSA were given byi.v. route once every two days for ten times. Finally, three mice pergroup were sacrificed and others were used for survival assay

In another experiment, murine TS/A mammary adenocarcinoma cells werefirst cultured in DMEM supplemented with 0% FBS, 2 mM L-glutamine, 100U/ml penicillin and 100 μg/ml streptomycin with or without fibrillarproteins such as rVP1 (0.1-0.2 μM) treatment in a humidified atmosphereof 95% air and 5% CO₂ at 37° C. After harvest, the TS/A cells pretreatedwith or without rVP1 were intravenously injected (3×10⁵/100 μlPBS/mouse) in the lateral tail vein of nine mice per group,respectively. After 14 days, all mice were sacrifice and harvested thelungs.

Establishment of Metastatic Xenograft Implantation in a Murine Model ofHuman Breast Cancer.

Human MDA-MB-231 mammary adenocarcinoma cells were cultured in DMEM/F12supplemented with 10% FBS, 2 mM L-glutamine, 100 U/ml penicillin and 100μg/ml streptomycin in a humidified atmosphere of 95% air and 5% CO₂ at37° C. After harvest, MDA-MB-231 cells (3×10⁵/100 μl PBS/mouse) wereintravenously injected into the lateral tail vein of nine mice pergroup. One day after tumor injection, fibrillar proteins such as F-HSA(1 mg/Kg BW) were given by i.v. route once every two days for ten timeswhile control group were injected with medium only. Three mice per groupwere sacrificed at this time point and the rest of mice were kept tomeasure the survival rate.

Establishment of Orthotropic Xenograft Implantation in a Murine Model ofHuman Prostate Cancer.

Human PC-3 prostate cancer cells were cultured in RPMI-1640 mediumsupplemented with 10% FBS, 2 mM L-glutamine, 100 U/ml penicillin and 100μg/ml streptomycin in a humidified atmosphere of 95% air and 5% CO₂ at37° C. After harvest, PC-3 cells were washed and orthotopically injectedin prostate (1×10⁵/20 μL PBS/mouse). In one group, four wks after PC-3implantation, fibrillar proteins such as rVP1 (25 mg/kg) was given byi.v. route, while in the other group rVP1 was given 10 wks after cancerimplantation. The same amount of rVP1 was administered three times perweek for 6 weeks and at the end of rVP1 treatment, 3 mice weresacrificed in each group to detect the cancer metastasis. The rest ofmice were kept to measure the survival rate.

Establishment of Metastatic Xenograft Implantation in a Murine Model ofHuman SK-OV-3 Ovarian Cancer.

Animal studies were performed in compliance with the guidelines for thecare and use of laboratory animals of the National Defense MedicalCenter, Taiwan. BALB/cAnN-Foxn1 female nude mice, 8 weeks old, obtainedfrom National Animal Center, Taiwan, were inoculated with 5×10⁶ SK-OV-3cancer cells per mouse by intraperitoneal injection 6 days after SK-OV-3implantation, mice were treated with fibrillar proteins (eg. rVP1, 15mg/Kg BW) or PBS (control) and the treatments repeated every other dayfor 60 days. The survival percentage and body weights were recordeduntil the mice died or 340 days after tumor implantation.

Isolation of SK-OV-3ip.1 Cells.

Mice were sacrificed by instant cervical dislocation 60 days afterSK-OV-3 cell implantation. Three ml of PBS was then injected to thebelly and the intraperitoneal cells were retrieved (about 2 ml) using a5-ml syringe and a 25 gauge×1″ needle. The cells were centrifuged at200×g for 5 min at 4° C. and the cell pellet collected. To remove redblood cells, 5 times cell pellet volume of NH₄Cl (0.144 M) and ½ timescell pellet volume of NH₄HCO₃ (0.01 M) were added and incubated at 4° C.for 5 min. The cells were centrifuged at 200×g for 5 min at 4° C. andthe cell pellet was collected. Cells were cultured in McCoy's 5A mediumwith 20% FBS in a 5% CO₂-humidified atmosphere at 37° C. for 3 daysbefore Western blot analysis of HER-2 receptor levels, using standardWestern blot techniques following resolution by SDS-PAGE on a 8-16%gradient gel (Invitrogen).

Histopathology.

The organs were fixed in 10% neutral buffered formalin. The tissues werefarther embedded in paraffin, cut at 4 μm sections, stained withhematoxylin and eosin (H&E) for light microscopy.

Statistical Analysis.

All data were expressed as means±standard error and estimated withMicrosoft Excel. For the survival data, the log-rank test was used todetermine differences between groups treated with or without drugs. TheStudent's t-test and ANOVA were performed to assess overall differencesbetween the different treatments. Values of P<0.05, P<0.01, and P<0.001were considered statistically significant.

-   -   rVP1 suppressed cell invasion and/or migration in MDA-MB-231        cells, PC-3 cells, 22Rv1 cells, SK-OV-3 cells, CaSki cells, and        SK-OV-3ip.1 cells in vitro

To investigate whether rVP1 suppressed tumor cell invasion and/ormigration, cell invasion and/or migration were measured using Boydenchamber assay. After various concentrations (0.1 μM to 0.2 μM rVP1 inhuman mammary adenocarcinoma cell line MDA-MB-231 cells, human prostateadenocarcinoma cell line PC-3 cells, human prostate carcinoma cell line22Rv1 cells; 0.2 μM to 0.4 μM rVP1 in human ovarian carcinoma cell lineSK-OV-3 cells and SK-OV-3ip.1 cells; 0.2 μM to 0.6 μM rVP1 in humancervical carcinoma cell line CaSki cells) of rVP1 treatment, cellinvasion and/or migration were significantly suppressed. Of note, theseconcentrations of rVP1 did not affect cell viability as determined byusing MTT assay or WST-1 assay (FIGS. 1-3). Therefore, rVP1 cansignificantly suppress metastasis of human breast cancer, prostatecancer, cervical cancer, and ovarian cancer cell lines in vitro.

Example 2 rVP1 Suppressed Tumor Cell Metastasis In Vivo

A. In Vitro rVP1-Treated MDA-MB-231 Cells Followed by i.v. Injectioninto BALB/C Mice.

We then examined whether pretreated MDA-MB-231 human mammaryadenocarcinoma cells with rVP1 for 24 h to inhibit metastatic ability invitro then injected the cells intravenously in mice could reduce theirmetastasis in mice lung as compare with MDA-MB-231 cells without rVP1pretreatment. Data showed that 0.1 and 0.2 μM rVP1-treated MDA-MB-231cells significantly decreased cancer cells metastasis in mice lungtissue (FIG. 4A). The related lung weight and the number of tumor fociin lung were also significantly decrease compared to tumor group withoutrVP1 treatment (FIGS. 4B-C).

B. Orthotropic PC-3 Xenograft Model.

We also examined whether rVP1 could suppress prostate cancer metastasisin vivo. PC-3 human prostate adenocarcinoma cells were firstorthotopically implanted to nude mice' prostate and 4-wks or 10-wksafter implantation, rVP1 (25 mg/kg) was then injected intravenouslythree times every week for 6 wks. At the end of rVP1 treatment, wesacrificed mice to undertake autopsy. Our data showed that rVP1significantly suppressed PC-3 cells metastasized to lymph nodes (Table1, FIG. 5A) and pelvic bones, and inhibited osteolysis in pelvic bones(FIG. 5B).

TABLE 1 The percentage and location of metastatic lymph nodes with rVP1treatment in PC-3-implanted nude mice. rVP1 (given rVP1 (given 4 wksafter 10 wks after tumor tumor Tumor implantation) Tumor implantationRenal 66.67% (2/3) 0% (0/3) 100% (3/3) 0% (0/3) nodes Lumbar 100% (3/3)0% (0/3) 100% (3/3) 0% (0/3) nodes Sacral 0% (0/3) 0% (0/3) 66.67% (2/3)0% (0/3) node Sciatic 0% (0/3) 0% (0/3) 66.67% (2/3) 0% (0/3) nodeInguinal 33.33% (1/3) 0% (0/3) 33.33% (1/3) 0% (0/3) node

C. Metastatic SK-OV-3 Xenograft Model.

Our in vitro data showed that rVP1 suppressed SK-OV-3 human ovariancarcinoma cell invasion. To examine whether rVP1 could also suppressSK-OV-3 ovarian cancer metastasis in vivo, metastatic xenograftimplantation in a murine model of human SK-OV-3 ovarian cancer wasestablished. We found that after 60 days, livers from PBS-treatedSK-OV-3 bearing mice were surrounded by tumor cells. In the SK-OV-3bearing mice injected with rVP1 (15 mg/kg) intraperitoneally every otherday for 60 days, on the other hand, we did not observe any apparenttumor invasion of their livers (FIG. 6).

Example 3 F-HSA Suppressed Tumor Cell Invasion and/or Migration inMDA-MB-231 Cells, PC-3 Cells, 22Rv1 Cells and CaSki Cells In Vitro

To examine whether other fibrillar proteins such as F-HSA alsosuppressed cancer cell invasion and/or migration, the effect of F-HSA oncancer cell invasion and/or migration were measured by using Boydenchamber assay. After being treated with various concentrations of F-HSA(0.1 μM to 0.2 μM F-HSA in MDA-MB-231 cells; 0.025 to 0.1 μM F-HSA inPC-3 cells; 0.025 to 0.05 μM F-HSA in 22Rv1 cells; 0.2 to 0.4 μM F-HSAin CaSki cells), the invasion and/or migration abilities of a variety ofcancer cells were significantly suppressed. It is of note that at theseconcentrations, F-HSA did not affect cell viability by using MTT assay(FIG. 7).

Example 4 F-HSA Suppressed Tumor Cell Metastasis In Vivo

To further examine whether F-HSA could suppress tumor cell metastasis invivo, murine mammary adenocarcinoma TS/A cells were intravenouslyinjected into the lateral tail vein of BALB/c mice or human mammaryadenocarcinoma MDA-MB-231 cells were injected into nude micerespectively. F-HSA (1 mg/kg) was then injected intravenously at nextday and then one time every two days for ten times. At the end of F-HSAtreatment, mice were sacrificed to detect the metastasis of lungs. Ourresults showed that F-HSA significantly suppressed the metastasis ofTS/A cells and MDA-MB-231 cells to lung (FIGS. 8A and 9A) as compared toTS/A or MDA-MB-231 bearing mice treated with control medium only. Therelative lung weight and the number of tumor foci in lung of F-HSAtreated TS/A or MDA-MB-231 bearing mice were significantly less thanthose of TS/A or MDA-MB-231 bearing mice without any drug treatment(FIGS. 8B-C and 9B-C).

Example 5 F-BSA Suppressed CaSki Cell Invasion In Vitro

We also examined whether other fibrillar proteins such as F-BSAsuppressed CaSki human cervical carcinoma cell invasion. Cell invasionwas measured by using Boyden chamber assay. We found that at 0.1 μM-0.2μM, although F-BSA did not affect cell viability as indicated by MTTassay, it significantly suppressed CaSki cell migration/invasion (FIG.10).

Example 6 F-HSA Exhibits Enhanced Fluorescence Levels ofAmyloid-Specific Dye ThT in a Dose-Dependent Manner

FIG. 12 shows an implementation of experimental data shows that F-HSA,like amyloid fibrils Aβ (1-42), exhibit enhanced fluorescence level ofamyloid-specific dye ThT in a dose-dependent manner as compared with BSAnot processed by the Superdex-200 column. This result shows that F-HSAhas a fibrillar structure like Aβ (1-42), whereas HSA has a globularstructure. (Binding to ThT is one of the characteristics of amyloid-likeproteins.)

Example 7 F-HSA has a Cytotoxic Effect on Breast Cancer Cells

FIG. 13 shows F-HSA's cytotoxic effect in TS/A cells and FIG. 14 showsF-HSA's cytotoxic effect in MDA-MB-231 cells. Each respective cell typewas treated for 16 h in serum-free culture medium with variousconcentrations of F-HSA. Cell viability was determined by the MTT assay.Globular HSA has no cytotoxic effect on normal or cancer cells.

Example 8 F-HSA has a Cytotoxic Effect on Ovarian and Cervical CancerCells

FIG. 15 shows F-HSA's cytotoxic effect in SKOV-3 cells and FIG. 16 showsF-HSA's cytotoxic effect in CaSki cells. Each respective cell type wastreated for 16 h in serum-free culture medium with variousconcentrations of F-HSA. Cell viability was determined by the MTT assay.

Example 9 F-HSA has a Cytotoxic Effect on Prostate Cancer Cells

FIG. 17 shows F-HSA's cytotoxic effect in PC-3 cells and FIG. 7 showsF-HSA's cytotoxic effect in 22 Rvl cells. The respective cell type wastreated for 16 h in serum-free culture medium with variousconcentrations of F-HSA. Cell viability was determined by the MTT assay.

Example 10 F-HSA has a Cytotoxic Effect on Lung Cancer Cell Lines

According to implementations shown in FIG. 19, F-HSA was shown to inducecytotoxicity in adenocarcinoma cell lines A549, CL1-0, Cl1-5, H1299,PC13, and PC14; squamous cell carcinoma lung cancer cell line H520; andlarge cell lung cancer carcinoma cell line H661. FIG. 19 shows the IC₅₀of each of the respective cell lines.

Example 11 F-HSA Suppresses Tumor Cell Invasion and Migration in Vitro

F-HSA was also shown to be effective in suppressing tumor cell invasionand migration in vitro, as shown according to implementations ofexperimental data in FIG. 20. As shown in FIGS. 9A, 9C, 9E, and 9G,F-HSA significantly reduced the tumor cells invasion/migrationabilities, at concentrations which did not affect viability of eithercancer or normal cells.

Example 12 F-HSA Suppressed Tumor Cell Metastasis in Vivo

F-HSA suppressed breast cancer tumor cell lines TS/A and MDA-MB-231 invivo. FIGS. 21A and 22A show F-HSA significantly suppressed themetastasis of breast cancer TS/A cells and MDA-MB-231 cells to lungcompared with TS/A or MDA-MB-231 bearing mice without F-HSA treatment.FIGS. 21B, 21C, 22B, and 22C measure the weight and the number of tumorcell foci in the lung tissues, which further confirmed the efficacy ofF-HSA in vivo.

According to implementations, breast cancer cells were injected via thetail vein of the subjects. Tumor cell foci detected in the lung tissueindicated that the breast cancer cells had metastasized into lung.

Materials and Methods

Preparation of F-HSA. Twenty milligrams of HSA was dissolved in 10 ml ofPBS with 1% SDS (w/v). The HSA solution was sonicated for 5 min andsubsequently applied to a Superdex-200, which was previouslyequilibrated with the eluting solution (25 mM Tris-HCl (pH 8.0), 1 mMEDTA, 0.1 M NaCl, and 0.05% SDS). The column was eluted at the rate of 1ml/min and fractions C3 to C7 that contained HSA were pooled. The pooledfractions were concentrated to 2-3 mg/ml then dialyzed against PBS withCellu-Sep T4/Nominal (MWCO: 12,000-14,000 Da) dialysis membrane. New PBSbuffer was exchanged every two hours at room temperature three times.The yield of the HSA-5200 was about 75%.

Thioflavin T (ThT) fluorescence assay. Binding to ThT is one of thecharacteristics of amyloid-like proteins. For fluorescence measurements,increasing concentrations of proteins were incubated with 20 μM ThT for1 h at room temperature, the fluorescence was then measured intriplicate on a Wallac Victor² 1420 Multilabel Counter (Perkin ElmerLife Science, Waltham, Mass., USA). Excitation and emission wavelengthswere 430 nm and 486 nm, respectively. ThT background signal from buffersolution was subtracted from corresponding measurements.

Cell survival was determined by MTT colorimetric assay. Exponentiallygrowing cells (2×10⁴ cells/well for TS/A) were seeded in a 96-well platein medium with 10% FBS and incubated for 24 h. Treatment of cells with aseries of concentrations of proteins was carried out in serum-freemedium for 16 hr indication at 37° C. After treatment, MTT solution wasadded to each well (0.5 mg/ml), followed by a 4 h incubation period. Theviable cell number is directly proportional to the production offormazan, which, following solubilization with isopropanol, can bemeasured spectrophotometrically at 570 nm by an ELISA plate reader.

Cell Viability Assays. Cell viability was measured by WST-1 assayaccording to the manufacturer's instructions (Roche, Mannheim, Germany).In brief, 2×10⁴ cells were added to 100 μl media per well on a 96 wellplate and incubated at 37° C. in 5% CO₂ overnight in a humidifiedincubator. The cells attached to the wells were incubated in serum-freemedium and treated with various concentrations of F-HSA. Afterincubation at 37° C. in 5% CO₂ for 16 h to allow the drug to takeeffect, 10 μl WST-1 reagent was added to each well. The plate was thenplaced onto a shaking table and shaken at 150 rpm for 1 min. Afterincubation at 37° C. in 5% CO₂ for another 2 h to allow the WST-1reagent to be metabolized, the proportion of surviving cells weredetermined by optical density (450 nm test wavelength, 690 nm referencewavelength). The percentage of surviving cells was calculated as (O.D.treatment/O.D. control)×100% while the percentage of growth inhibitionwas calculated as [1−(O.D. treatment/O.D. control)]×100%. According tothis experiment, IC₅₀ is the concentration at which the reagent yields50% inhibition of the cellular viability.

Cell Migration and Invasion Assays. Cell migration and invasion weredetermined by using Boyden chamber migration and invasion assay(Corning). In brief, the 8-μm pore membranes of the upper chambers werecoated with 20 μg/ml fibronectin (for cell migration assay) or 40 μg/mlMatrigel (for cell invasion assay) and placed in a well with 1 ml of PBSand incubated for 2 h at 37° C. Cancer cells (1×10⁵) in 100 μl of serumfree culture medium were seeded in the upper chamber for 1 h. A seriallydiluted concentration of F-HSA was added into the upper chamber and thenthe culture medium containing 10% FBS was added to the lower chamber.Cells were incubated for 24 h at 37° C. After incubation, cells on theupper side of the membrane were removed by wiping it with a cotton swab,and cells that had migrated onto the lower membrane surface weredissociated by using cell dissociation solution (Sigma) and counted byflow cytometer (BD company). At these concentrations, however, F-HSA didnot affect cell viability when measured with the MTT assay, and as shownin FIGS. 9B, 9D, 9F, and 9H. Viability and cytotoxicity was measuredusing MTT or WST-1 assay. These kits are designed for thespectrophotometric measurement of cell growth as a function ofmitochondrial activity in living cells (Roche).

Breast Cancer Cell Metastasis In Vivo. TS/A murine mammaryadenocarcinoma cells were intravenously injected into the lateral tailvein of BALB/c mice or MDA-MB-231 human mammary adenocarcinoma cellswere injected into nude mice. F-HSA (1 mg/kg) was then injectedintravenously at next day and then one time every two days for tentimes. At the end of F-HSA treatment, mice were sacrificed to detect themetastasis of lungs.

1-41. (canceled)
 42. A pharmaceutical composition for suppressing cancermetastasis in a subject comprising a fibrillar protein and apharmaceutically acceptable excipient.
 43. The composition of claim 42,wherein said fibrillar protein comprises fibrillar albumin.
 44. Thecomposition of claim 42, wherein said fibrillar protein comprisesfibrillar human serum albumin.
 45. The composition of claim 42, whereinsaid fibrillar protein comprises capsid proteins of thefoot-and-mouth-disease virus.
 46. A pharmaceutical compositioncomprising a therapeutically effective amount of fibrillar human serumalbumin and a pharmaceutically acceptable carrier for use in treating amammal having cancer.
 47. The pharmaceutical composition of claim 46,wherein said cancer is characterized by overexpression of α5β1 and/orαvβ3 integrin.
 48. A method of manufacturing the composition accordingto claim 44, comprising the following steps: manufacturing fibrillarhuman serum albumin; and mixing the fibrillar human serum albumin in atherapeutically effective amount with a pharmaceutically acceptablecarrier.
 49. The method of claim 48, wherein the step of manufacturingthe fibrillar human serum albumin comprises the following steps:dissolving human serum albumin in a detergent solution; applying thedissolved human serum albumin through a gel filtration column with apore size to separate proteins of 70 kDa molecular weight and above; andeluting the human serum albumin from the column.
 50. A method oftreating cancer in a human patient comprising administering to saidpatient the composition of claim
 42. 51. A method of treating cancercomprising administering to a subject in need thereof an effectiveamount of a fibrillar protein and a pharmaceutically acceptable carrier,wherein said method reduces cancer metastasis in said subject.
 52. Themethod according to claim 51, wherein said fibrillar protein comprisesfibrillar albumin.
 53. The method according to claim 52, wherein saidfibrillar protein comprises fibrillar human serum albumin.
 54. Themethod according to claim 51, wherein said fibrillar protein comprisesfibrillar capsid proteins of the foot-and-mouth-disease virus.
 55. Themethod of claim 51, wherein said fibrillar protein is a chimericprotein.
 56. The method of claim 51, wherein said subject is a human.57. The method of claim 51, wherein said cancer is characterized byoverexpression of α5β1 and/or αvβ3 integrin.
 58. The method of claim 51,wherein said administering is selected from the group consisting ofintravenous injection, subcutaneous injection, intraperitonealinjection, intraarterial injection, intramuscular injection,intralesional injection into the tumor, intralesional injection adjacentto the tumor, intravenous infusion, and intraarterial infusion.
 59. Themethod of claim 51, wherein said method further comprises administeringa second therapeutic agent and/or providing radiation therapy to saidsubject.
 60. The method of claim 59, wherein said second therapeuticagent is a chemotherapeutic agent.
 61. The method of claim 51, whereinsaid fibrillar protein is produced by a method comprising the followingsteps: dissolving a protein in a detergent solution to provide adissolved protein; applying said dissolved protein through a gelfiltration column with a pore size that can separate proteins of 70 kDamolecular weight and above; eluting said dissolved protein from said gelfiltration column to provide an eluate; and removing said detergent fromsaid eluate.